METHODS AND COMPOSITIONS FOR cDNA SYNTHESIS AND SINGLE-CELL TRANSCRIPTOME PROFILING USING TEMPLATE SWITCHING REACTION

ABSTRACT

This application discloses methods for cDNA synthesis with improved reverse transcription, template switching and preamplification to increase both yield and average length of cDNA libraries generated from individual cells. The new methods include exchanging a single nucleoside residue for a locked nucleic acid (LNA) at the TSO 3′ end, using a methyl group donor, and/or a MgCl 2  concentration higher than conventionally used. Single-cell transcriptome analyses incorporating these differences have full-length coverage, improved sensitivity and accuracy, have less bias and are more amendable to cost-effective automation. The invention also provides cDNA molecules comprising a locked nucleic acid at the 3′-end, compositions and cDNA libraries comprising these cDNA molecules, and methods for single-cell transcriptome profiling.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/912,556, filed Feb. 17, 2016, which is the U.S. nationalphase application of International Patent Application No.PCT/US2014/052233, filed Aug. 22, 2014, which in turn claims priority toU.S. Provisional Patent Application Ser. No. 61/869,220, filed Aug. 23,2013, the contents of which are hereby incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to methods for synthesis of doublestranded cDNA with improved yield and average length, and cDNA moleculessynthesized and cDNA libraries generated from individual cells.

BACKGROUND OF THE INVENTION

Single-cell gene expression analyses hold promise to characterizecellular heterogeneity, but current methods sacrifice either thecoverage, sensitivity or throughput. Several methods exist forfull-length cDNA construction from large amounts of RNA, including capenrichment procedures (Maruyama, K. & Sugano, S., Gene 138, 171-174(1994); Carninci, P. & Hayashizaki, Y., Meth. Enzymol. 303, 19-44(1999); Das, M., et al., Physiol. Genomics 6, 57-80 (2001)), but it isstill challenging to obtain full-length coverage from single-cellamounts of RNA. Existing methods use either 3′ end polyA-tailing of cDNA(e.g., Tang, F. et al., Nat Methods 6, 377-382 (2009); Sasagawa, Y. etal., Genome Biol. 14, R31 (2013)) or template switching (Zhu, Y. Y., etal., Bio Techniques 30, 892-897 (2001); Ramsköld, D. et al., NatBiotechnol. 30, 777-782 (2012)), whereas other methods sacrificefull-length coverage altogether for early multiplexing (Islam, S. etal., Genome Res. (2011). doi:10.1101/gr.110882.110; Hashimshony, T., etal., Cell Rep. 2, 666-673 (2012)). It has recently been shown thatSmart-Seq, which relies on template switching, has more even readcoverage across transcripts than polyA-tailing methods (Ramsköld, D. etal., Nat. Biotechnol. 30, 777-782 (2012)), consistent with the commonuse of template switching in applications designed to directly captureRNA 5′ ends, including nanoCAGE (Plessy, C. et al., Nat Methods 7,528-534 (2010)) and STRT (Islam, S. et al., Genome Res. (2011).doi:10.1101/gr.110882.110). Single-cell applications utilizing templateswitching are dependent upon the efficiency of the reversetranscription, the template switching reaction, and a uniform polymerasechain reaction (PCR) preamplification to obtain representative cDNA insufficient amounts for sequencing. Despite the widespread use of thesereactions, no systematic efforts to improve cDNA library yield andaverage length from single-cell amounts have been reported.

SUMMARY OF THE INVENTION

The present invention provides improved methods for synthesis of cDNA,in particular, in the reverse transcription, template switching andpreamplification of single cell applications utilizing templateswitching reactions, to increase both yield and average length of cDNAlibraries generated from individual cells. Single-cell transcriptomeanalyses incorporating these differences have improved sensitivity andaccuracy, and are less biased and more amenable to cost-effectiveautomation.

Specifically, to improve full-length transcriptome profiling from singlecells, this application discloses evaluation of a large number ofvariations to reverse transcription, template switching oligonucleotides(TSO) and PCR preamplification, and comparison of the results tocommercial Smart-Seq (hereafter called SMARTer®) in terms of cDNAlibrary yield and length. The modifications disclosed hereinsurprisingly and significantly increased both the yield and length ofthe cDNA obtained from as little as 1 ng of starting total RNA.

In one embodiment, the present invention provides a method for preparingDNA that is complementary to an RNA molecule, the method comprisingconducting a reverse transcription reaction in the presence of atemplate switching oligonucleotide (TSO) comprising a locked nucleicacid residue.

In another embodiment, the present invention provides a method ofincreasing the yield of cDNA, comprising use of an additive, such as amethyl group donor, in the cDNA synthesis. In one embodiment, the methylgroup donor is betaine.

In another embodiment, the present invention provides a method ofincreasing the yield of cDNA, comprising use of an increasedconcentration of metal salt, for example, MgCl₂, in the synthesis ofcDNA.

In a preferred embodiment, the method comprises use of a methyl groupdonor in combination with an increased concentration of MgCl₂ in thecDNA synthesis. In a particularly preferred embodiment, the methodcomprises use of methyl group donor betaine in combination with anincreased concentration of MgCl₂, which has shown a significant positiveeffect on the yields of cDNA.

In another embodiment, the present invention provides a method ofincreasing the average length of a preamplified cDNA, comprisingadministering dNTPs prior to the RNA denaturation rather than in thereverse transcriptase (RT) master mix.

In another embodiment, the present invention provides a cDNA libraryproduced by a method according to any of the embodiments disclosedherein.

In another embodiment, the present invention provides use of a cDNAlibrary produced according to any of the embodiments disclosed hereinfor single-cell transcriptome profiling.

In another embodiment, the present invention provides a method foranalyzing gene expression in a plurality of single cells, comprising thesteps of preparing a cDNA library according to a method according to anyembodiment disclosed herein; and sequencing the cDNA library.

It has been demonstrated in accordance with the present invention thatthese methods performed on purified RNA are applicable to individualmetazoan cells, including for example mammalian cells.

In another embodiment, the present invention provides a templateswitching oligonucleotide (TSO) comprising an LNA at its 3′-end.

In another embodiment, the present invention provides use of a TSOaccording to any of the embodiments disclosed herein for synthesis ofdouble stranded cDNA.

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates improvements in cDNA library yield and length. (A)Median yield of preamplified cDNA from 1 ng total RNA using differenttemplate switching oligonucleotides, relative to those obtained usingthe rG3 oligo. All oligo sequences are found in Table 1. (B) Medianyield of preamplified cDNA from 1 ng total RNA in reactions with betaine(black) or without (gray) and as a function of increasing Mg²⁺concentration, relative to cDNA yields obtained using SMARTer®-likeconditions (betaine and 6 mM Mg²⁺). (C) Length of preamplified cDNAgenerated from 1 ng total mouse brain RNA in reactions that deployeddNTPs prior to RNA denaturation (early) or in RT master mix (late). (D)Median yield of preamplified cDNA from HEK293T cells using the LNA-G andSMARTer® IIA template switching oligos with the optimized protocol.Dashed lines indicate median yield from commercial SMARTer® reactions.(E) Median yield of preamplified cDNA from DG-75 cells in reactions withor without betaine. (F) Lengths of cDNA libraries generated from singleHEK293T cells in reactions with or without bead extraction. Note thatbrain mRNAs are naturally longer than cell line mRNAs. (A-F) Thereplicate measurements are represented as boxplots with the numbers ofreplicates per condition indicated in parenthesis. Significantdifferences in mean yield or length were determined using the Student'st-test.

FIG. 2 illustrates sensitive full-length transcriptome profiling insingle cells. (A) Percentage of genes reproducibly detected in replicatecells, binned according to expression level. All pair-wise comparisonswere performed within replicates for the optimized protocol and SMARTer®and reported as the mean and 90% confidence interval. (B) Standarddeviation in gene expression estimates within replicates in bins ofgenes sorted according to expression levels. Error bars, s.e.m. (n≥4).(C) The mean numbers of genes detected in HEK293T cells using SMARTer®and optimized protocol, at different RPKM cut-offs. Significant increasein gene detection in the optimized protocol was obtained at all RPKMthresholds (all with p<0.5; Student's t-test). (D) The mean fraction ofgenes detected as expressed (RPKM>1) in bins of genes sorted accordingto their GC content. The mRNA-Seq data from a human tissue was includedas a no-preamplification control. Error bars denote SEM (n≥4), and thelower panel shows the GC range for genes in each bin. (E) The meanfraction of reads aligning to the 3′ most 20% of the genes, 5′ most 20%and the middle 60% for single-cell data generated using differentprotocols. (F) Principal component analyses of single-cell geneexpression data showing the two most significant components. Cells arecolored according to preamplification enzyme and protocol variant.

FIG. 3 shows cDNA yields using LNA bases in template switchingoligonucleotides.

FIG. 4 illustrates single-cell RNA-Seq sensitivity and variability. (A)Percentage of genes reproducibly detected in replicate cells. (B)Standard deviation in gene expression estimates.

FIG. 5 illustrates validation of single-cell RNA-Seq results usinganother analysis pipeline. (A) Percentage of genes reproducibly detectedin replicate cells. (B) Standard deviation in gene expression estimates.(C) The mean numbers of genes detected. (D) The mean fraction of genesdetected as expressed. (E) The mean fraction of reads aligning to the 3′most 20% of the genes, 5′ most 20% and the middle 60% for single-celldata generated using different protocols.

FIG. 6 illustrates comparison of single-cell transcriptomic datagenerated with Smart-Seq2, Quartz-Seq and SMARTer. (A) Percentage ofgenes reproducibly detected in replicate cells. All pair-wisecomparisons were performed with Smart-Seq2 and Quartz-seq, and reportedas the mean and 90% confidence interval. (B) Standard deviation in geneexpression estimates in (A). (C) Percentage of genes reproduciblydetected in replicate cells. All pair-wise comparisons were performedwith Smart-Seq2 and SMARTer®, and reported as the mean and 90%confidence interval. (D) Standard deviation in gene expression estimatesin (C). (E) Percentage of genes reproducibly detected in replicatecells. All pair-wise comparisons were performed with Quartz-seq, andreported as the mean and 90% confidence interval. (F) Standard deviationin gene expression estimates in (E).

FIG. 7 illustrates mapping statistics for single-cell librariesgenerated using SMARTer, optimized Smart-Seq and variants of theoptimized protocol. (A) Fraction of uniquely aligned reads with 1 to 9mismatches for each single-cell RNA-Seq library. (B) Percentage of readsthat aligned uniquely, aligned to multiple genomic coordinates, or didnot align for all single-cell RNA-Seq libraries. (C) Fraction ofuniquely aligned reads that mapped to exonic, intronic or intergenicregions. (D) Number of sequenced reads per cell and library preparationprotocol.

FIG. 8 illustrates gene expression and GC levels in single-cell RNA-Seqprotocols.

FIG. 9 illustrates single-cell RNA-Seq sensitivity and variability. (A)Percentage of genes reproducibly detected in replicate cells. (B)Standard deviation in gene expression estimates.

FIG. 10 illustrates read coverage across genes in single-cell RNA-Seqdata.

FIG. 11 illustrates read coverage across transcripts. (A) Mean fractioncoverage read for all genes. (B)-(F) Transcripts grouped by length into5 equal-sized bins.

FIG. 12 illustrates read peaks in single-cell RNA-Seq data. (A) Numberof genes with one or more high density peaks per single-cell RNA-Seqlibrary. (B) Heatmaps of read densities across genes with peaks in thehighest number of libraries.

FIG. 13 illustrates assessment of the technical and biologicalvariability in single-cell transcriptomics using Smart-Seq2. (A)Percentage of genes reproducibly detected in dilutions of HEK cells. (B)Standard deviation in gene expression estimates for (A). (C) Percentageof genes reproducibly detected in dilutions of HEK total RNA. (D)Standard deviation in gene expression estimates for (D). (E) Standarddeviation in gene expression estimates for (D) with pair-wisecomparisons of individual cells.

FIG. 14 illustrates comparison of libraries generated with commercialTn5 (Nextera) to in-house produced Tn5. (A) Percentage of genesreproducibly detected using in-house conditions. (B) Standard deviationin gene expression estimates for (A). (C) Percentage of genesreproducibly detected using commercial buffers and conditions. (D)Standard deviation in gene expression estimates for (D). (E) Differencesin reactions carried out in (A).

DETAILED DESCRIPTION OF THE INVENTION

This application discloses methods for cDNA synthesis with improvedreverse transcription, template switching and preamplification toincrease both yield and average length of cDNA libraries generated fromindividual cells.

In one embodiment, the present invention provides a method for preparingDNA that is complementary to an RNA molecule, comprising the steps of:

annealing a cDNA synthesis primer to the RNA molecule and synthesizing afirst cDNA strand to form an RNA-cDNA intermediate; and

conducting a reverse transcriptase reaction by contacting the RNA-cDNAintermediate with a template switching oligonucleotide (TSO), whereinthe TSO comprises a locked nucleic acid (LNA) at its 3′-end, underconditions suitable for extension of the first DNA strand that iscomplementary to the RNA molecule, rendering it additionallycomplementary to the TSO.

In another embodiment of the present invention, the reversetranscription reaction is conducted in the presence of a methyl groupdonor and a metal salt.

In another embodiment of the present invention, the methyl group donoris betaine.

In another embodiment of the present invention, the metal salt is amagnesium salt.

In another embodiment of the present invention, the magnesium salt has aconcentration of at least 7 mM, at least 8 mM, or at least 9 mM.

In another embodiment of the present invention, the template switchingoligonucleotide optionally comprises one or two ribonucleotide residues.

In another embodiment of the present invention, the template switchingoligonucleotide comprises at least one or two ribonucleotide residuesand an LNA residue.

In another embodiment of the present invention, the at least one or tworibonucleotide residues are riboguanine.

In another embodiment of the present invention, the locked nucleic acidresidue is selected from the group consisting of locked guanine, lockedadenine, locked uracil, locked thymine, locked cytosine, and locked5-methylcytosine.

In another embodiment of the present invention, the locked nucleic acidresidue is locked guanine.

In another embodiment of the present invention, the locked nucleic acidresidue is at the 3′-most position.

In another embodiment of the present invention, the template switchingoligonucleotide comprises at the 3′-end two ribonucleotide residues andone locked nucleotide residue characterized by formula rGrG+N, wherein+N represents a locked nucleotide residue.

In another embodiment of the present invention, the template switchingoligonucleotide comprises rGrG+G.

In another embodiment of the present invention, the methyl group donoris betaine, and the metal salt is MgCl₂ at a concentration of at least 9mM.

In another embodiment of the present invention, the method furthercomprises amplifying the DNA strand that is complementary to the RNAmolecule and the template switching oligonucleotide using anoligonucleotide primer.

In another embodiment of the present invention, the template switchingoligonucleotide is selected from the oligonucleotides in Table S2.

In another embodiment of the present invention, the cDNA synthesisprimer is an oligo-dT primer.

In another embodiment of the present invention, the cDNA is synthesizedon beads comprising an anchored oligo-dT primer.

In another embodiment of the present invention, the oligo-dT primercomprises a sequence of 5′-AAGCAGTGGTATCAACGCAGAGTACT₃₀VN-3′ (SEQ ID NO:1), wherein “N” is any nucleoside base, and “V” is selected from thegroup consisting of “A”, “C” and “G”.

In another embodiment of the present invention, the method furthercomprises PCR preamplification, tagmentation, and final PCRamplification.

In another embodiment of the present invention, the PCR preamplificationis conducted without purifying the cDNA obtained from reversetranscription reaction.

In another embodiment of the present invention, the RNA is total RNA ina cell.

In another embodiment, the present invention provides a cDNA libraryproduced by the method according to any embodiment disclosed herein.

In another embodiment, the present invention provides use of a cDNAlibrary produced by the method according to any embodiment disclosedherein for single-cell transcriptome profiling.

In another embodiment, the present invention provides a method foranalyzing gene expression in a plurality of single cells, the methodcomprising the steps of: preparing a cDNA library produced by the methodaccording to any embodiment disclosed herein; and sequencing the cDNAlibrary.

In another embodiment, the present invention provides a templateswitching oligonucleotide (TSO) comprising a locked nucleotide residueat the 3′-end. The TSOs of the present invention can be used in thesynthesis of cDNA to improve yield and length.

In another embodiment, the TSO comprises three nucleotide residues atthe 3′-end, wherein said three nucleotide residues are selected from thegroup consisting of +N+N+N, N+N+N, NN+N, rN+N+N, and rNrN+N, wherein Nat each occurrence is independently a deoxyribonucleotide residue, rN ateach occurrence is independently a ribonucleotide residue, and +N ateach occurrence is independently a locked nucleotide residue.

In one embodiment, the portion of the TSO that is on the 5′ side of thethree nucleotide residues at the 3′-end, also referred to herein as the5′-portion, comprises an arbitrary nucleotide sequence comprised ofribonucleotides, deoxyribonucleotides, or mixtures thereof. In onepreferred embodiment, the 5′-portion of the TSO comprises allribonucleotides. In another preferred embodiment, the 5′-portion of theTSO comprises all deoxyribonucleotides.

In another embodiment, the locked nucleotide residue in the TSOs isselected from the group consisting of locked guanine, locked adenine,locked uracil, locked thymine, locked cytosine, and locked5-methylcytosine

In another embodiment, the three nucleotide residues at the 3′-end ofthe TSOs are NN+G or rNrN+G, wherein N at each occurrence isindependently a deoxyribonucleotide residue, and rN at each occurrenceis independently a ribonucleotide residue.

In another embodiment, the three nucleotide residues at the 3′-end ofthe TSOs are rGrG+N, wherein +N is locked nucleotide residue.

In another embodiment, the three nucleotide residues at the 3′-end ofthe TSOs are rGrG+G.

The TSOs preferably have a length of from about 10 to about 50nucleotides, or from about 15 to about 45 nucleotides, or from about 20to about 40 nucleotides, or from about 24 to about 35 nucleotides, orabout 30 nucleotides.

In another embodiment, the present invention provides use of a TSOaccording to any one of the embodiments disclosed herein in thesynthesis of a cDNA.

Examples of metal cations useful for the present invention include, butare not limited to, Mg²⁺ and Mn²⁺, with Mg²⁺ preferred; and theirconcentrations can be in the range of 0-30 μM, inclusive, with apreferred range of 3-20 μM, and a more preferred range of 9-12 μM.

In addition to methyl donor betaine, other additives that may be addedin the cDNA synthesis of the present invention include, but are notlimited to, trehalose, sucrose, glucose, maltose, DMSO (dimethylsulfoxide), formamide, non-ionic detergents, TMAC (tetramethylammoniumchloride), 7-deaza-2′-deoxyguanosine (dC⁷GTP), bovine serum albumin(BSA), and T4 gene 32 protein.

The present invention is applicable to reactions using all reversetranscriptases that are MMLV-related and have template switchingactivity. MMLV-related reverse transcriptases include wild-type Moloneymurine leukemia virus and its variants, including for examplederivatives lacking RNase H activity such as SUPER-SCRIPT II(Invitrogen), POWER SCRIPT (BD Biosciences) and SMART SCRIBE (Clontech).TSOs useful for the present invention may comprise barcodes, includingbut not limited to molecular barcodes or sample barcodes.

The cDNA synthesized according to the present invention may haveapplications as cDNA synthesized according to any literature methods,including but not limited to construction of small quantity cDNAlibrary, single-cell cDNA analyses, single-cell gene expressionanalyses, few-cell cDNA analyses, few-cell gene expression analyses,single-cell qPCR analyses (that use this preamplification step), and capcapturing based amplification.

The following non-limiting examples illustrate certain aspects of thepresent invention.

EXAMPLES Example 1 Methods Experiments Using Total RNA

RNA experiments were performed using the Control Total RNA supplied withthe SMARTer® Ultra Low RNA Kit for Illumina Sequencing (Clontech),extracted from mouse brain. One microliter of a 1 ng/μ1 solution wasused in each experiment and mixed with 1 μl of anchored oligo-dT primer(10 mM, AAGCAGTGGTATCAACGCAGAGTACT₃₀VN-3′ (SEQ ID NO: 1), where “N” isany base and “V” is either “A”, “C” or “G”) and 1 μl of dNTP mix (10 mM,Fermentas), denaturated at 72° C. for 3 min and immediately placed onice afterwards. Seven μl of the first strand reaction mix containing0.50 μl SuperScript II RT (200 U ml-1, Invitrogen), 0.25 μl RNAseinhibitor (20 U ml-1, Clontech), 2 μl Superscript II First-Strand Buffer(5×, Invitrogen), 0.25 μl DTT (100 mM, Invitrogen), 2 μl betaine (5 M,Sigma), 0.9 μl MgCl₂ (100 mM, Sigma), 1 μl TSO (10 μM, the complete listof the oligos can be found in Table S1) and 0.1 μl Nuclease-free water(Gibco) were added to each sample. Reverse transcription reaction wascarried out by incubating at 42° C. for 90 min, followed by 10 cycles of(50° C. for 2 min, 42° C. for 2 min). Finally, the RT was inactivated byincubation at 70° C. for 15 min.

PCR Pre-Amplification

In the original Smart-Seq protocol purification with Ampure XP beads isperformed after first strand cDNA synthesis. PCR is then carried outdirectly on the cDNA immobilized on the beads, after adding 2 μlAdvantage 2 Polymerase Mix (50×, Clontech), 5 μl Advantage 2 PCR Buffer(10×, Clontech), 2 μl dNTP mix (10 mM, Clontech), 2 μl IS PCR primer (12μM, Clontech) and 39 μl nuclease-free water to a final reaction volumeof 50 μl. In the present examples the cDNA was not purified after RT butjust added the same PCR master mix, taking into account that the volumeafter first strand cDNA synthesis is 10 μl and adjusting the amount ofwater accordingly. Reaction was incubated at 95° C. 1 min, then cycled15 times between (95° C. 15 sec, 65° C. 30 sec, 68° C. 6 min), with afinal extension at 72° C. for 10 min.

A second modification that significantly improved cDNA yield was thereplacement of Advantage 2 Polymerase mix with KAPA HiFi HotStartReadyMix (KAPA Biosystems). Purification after first strand cDNAsynthesis was omitted also in this case. The PCR master mix had thefollowing composition: 25 μl KAPA HiFi HotStart ReadyMix (2×, KAPABiosystems), 1 μl IS PCR primers (10 mM, 5′-AAGCAGTGGTATCAACGCAGAGT-3′(SEQ ID NO: 2)) and 14 μl nuclease-free water (Gibco). The program usedwas as follows: 98° C. 3 min, then 15 cycles of (98° C. 15 sec, 67° C.20 sec, 72° C. 6 min), with a final extension at 72° C. for 5 min.

Regardless of the PCR protocol used, PCR was purified using a 1:1 ratioof AMPure XP beads (Beckman Coulter), performing the final elution in 15μl of EB solution (Qiagen). Library size distribution was checked on aHigh-Sensitivity DNA chip (Agilent Bioanalyzer) after a 1:5 dilution.The expected average size should be around 1.5-2.0 kb and the fractionof fragments below 300 bp should be negligible. To evaluate theperformance of the different modifications introduced in the protocol,the amount of cDNA comprised in the interval 300-9000 bp in the AgilentBioanalyzer plot was assessed.

Tagmentation Reaction and Final PCR Amplification

Five nanograms of cDNA were then used for the tagmentation reactioncarried out with Nextera® DNA Sample Preparation kit (Illumina), adding25 μl of 2× Tagment DNA Buffer and 5 μl of Tagment DNA Enzyme, in afinal volume of 50 μl. Tagmentation reaction was incubated at 55° C. for5 min, followed by purification with DNA Clean & Concentrator™5 kit(Zymo Research) with a final elution in 20 μl Resuspension Buffer (RSB)from the Nextera® kit. The whole volume was then used for limited-cycleenrichment PCR, along with 15 μl of Nextera® PCR Primer Mix (NPM), 5 μlof Index 1 primers (N7xx), 5 μl of Index 2 primers (N5xx) and 5 μl ofPCR Primer Cocktail (PPC). A second amplification round was performed asfollows: 72° C. 3 min, 98° C. 30 sec, then 5 cycles of (98° C. 10 sec,63° C. 30 sec, 72° C. 3 min). Purification was done with a 1:1 ratio ofAMPure XP beads and samples were loaded on a High-Sensitivity DNA chipto check the quality of the library, while quantification was done withQubit High-Sensitivity DNA kit (Invitrogen). Libraries were diluted to afinal concentration of 2 nM, pooled and sequenced on Illumina HiSeq2000.

Single-Cell cDNA Isolation

Single HEK293T (human), DG-75 (human), C2C12 (mouse) and MEF (mouse)cells were manually picked under the microscope after resuspension inPBS. Volume of liquid was kept as low as possible, usually below 0.5 μland preferably below 0.3 μl. Cells were then transferred to a 0.2 mlthin-wall PCR tube containing 2 μl of a mild hypotonic lysis buffercomposed of 0.2% Triton X-100 (Sigma) and 2 UM of RNAse inhibitor(Clontech). Cells already picked were kept on ice throughout the processor stored at −80° C. if not used immediately. All the downstream stepswere the same as when using total RNA (see above), with the onlyexception of the quality control with the High Sensitivity DNA chip,where samples were loaded pure (without dilution), due to the limitedamount of cDNA obtained from RT in single cells.

When working with total RNA it was observed that cDNA yield could beincreased using a double amount of TSO or different combinations of TSOsand PCR enzymes (data not shown). To validate this finding, someexperiments on HEK293T cells were repeated using different amounts ofTSO (1 or 2 μl of a 10 μM solution), TSO types (rGrGrG, rGrG+G orrGrG+N) or PCR enzymes (KAPA HiFi or Advantage 2). Sequencing resultsfor the most significant comparisons are reported in Figures S2-S7. Thefinal protocol (i.e. “optimized”) refers to the one using only 1 μl ofthe 10 μM rGrG+G TSO and KAPA HiFi HotStart ReadyMix as enzyme in thefirst PCR (without AMPure XP bead purification).

Smart-Seq Experiments

To evaluate and compare the performance of the present method, cDNAlibraries were generated with the same total RNA and single cells usingthe Smart-Seq protocol, following manufacturer's instructions (seeClontech manual). After PCR pre-amplification, 5 ng of cDNA were usedfor the tagmentation reaction and processed exactly in the same way asdescribed above.

Statistical Analyses of cDNA Yield and Length

Performances of the different protocols were evaluated with regard tocDNA yield and average cDNA length according to the Bioanalyzer in therange of 3009,000 bp. For mouse brain total RNA samples, eachexperimental variable was evaluated in a pairwise manner selecting a setof experiments where all other variables are identical. Within that setof experiments, the significance for a change in yield or length,between the two variables, was evaluated using Student's t-test andWilcoxon rank sum test (Table 1, sheet B).

In the HEK293T cell experiments each optimized experimental setting wascompared to each other, as well as to the SMARTer® protocol, usingStudent's t-test and Wilcoxon rank sum test (Table 3, sheet B). Allanalyses and figures were produced with using R.

Read Alignments and Gene Expression Estimation

Single-cell libraries were sequenced with Nextera dual indexes (i7+i5)on an Illumina HiSeq 2000, giving 43 bp insert reads afterdemultiplexing and removing cellular barcodes. The reads were aligned tohuman (hg19) or mouse (mm10) genomes using STAR v2.2.0 (Dobin et al.Bioinformatics 2013 29(1): 15-21) with default settings and filtered foruniquely mapping reads. Gene expression values were calculated as RPKMvalues for each transcript in Ensembl release 69 and RefSeq (February2013) using rpkmforgenes (Ramsköld et al. PLoS Comp Biol., 5, e1000598,2009).

Single-Cell RNA-Seq Sensitivity and Variability

Analyses of gene detection in single HEK293T cells (FIG. 2A, FIGS. 5A,and 7A) were calculated over all possible pairs of technical replicatesfrom each experimental setting. Genes were binned by expression level inthe two samples, and were considered detected if the RPKM was above 0.1in both samples. The mean for all possible pairs of technical replicateswithin a group was used together with standard deviation using theadjusted Wald method. Analyses of variation (FIG. 2B and FIGS. 5B & 7B)were also calculated on pairs of samples, binning genes by the mean oflog expression, excluding genes below 0.1 RPKM in either sample. As geneexpression levels across single cells are often log normally distributed(Bengtsson Genome Res 2005 15(10): 1388-1392), absolute difference inlog₁₀ expression values and s.d. were calculated by multiplying meandifference in a bin with 0.886.

Analyses of Read Coverage and GC Tolerance

Gene body coverage was calculated using the RSeQC-2.3.4 package (Wang,Wang and Li. Bioinformatics 2012; 28(16):2184-5) for the longesttranscript of all protein coding genes (FIG. 2E and FIG. 8). Genedetection at different GC-content was calculated using longesttranscript for all protein coding RefSeq genes that were binned byGC-content into 10 equal sized bins, and the numbers of genes with nodetection, or detection at different RPKM cutoffs were calculated (FIG.2D and FIG. 6).

Read Peak Analyses

Some genes displayed unexplained peaks with high density of reads withinthe gene body. To identify these regions, the gene bodies of each genewere divided into 101 equally sized bins and each gene with at least onebin with >5 standard deviation read density over the mean readdistribution within that gene. In these analyses genes with lowexpressed genes (those with fewer reads than around 2,000-10,000 readsdepending on the sequencing depth per cell) were discarded. The numberof such genes in each cell is represented in FIG. 9A And the genes withpeaks in the highest number of HEK239T cells are displayed as heatmapsin FIG. 9B illustrating that the peaks are consistently found at thesame position in all experiments.

Example 2

To improve full-length transcriptome profiling from single cells, alarge number of variations to reverse transcription, template switchingoligonucleotide (TSO) and PCR preamplification (in total 457experiments) were evaluated, and the results were compared to commercialSmart-Seq (hereafter called SMARTer®) in terms of cDNA library yield andlength (Table 1). Importantly, modifications were identified thatsignificantly increased both cDNA yield and length obtained from 1 ng ofstarting total RNA (Table 1).

In particular, exchanging only a single guanylate for a locked nucleicacid (LNA) guanylate at the TSO 3′ end (rGrG+G), led to a 2-foldincrease in cDNA yield relative to the SMARTer® IIA oligo used incommercial Smart-Seq (p=0.003, Student's t-test; FIG. 1a , Table 2 andFIG. 3).

Additionally, it was discovered that the methyl group donor betaine incombination with higher MgCl₂ concentrations had a significant positiveeffect on yield (2-4 fold increase, p=0.0012, Student's t-test, for allcomparisons) (FIG. 1b ). The commercial Smart-Seq buffer has a finalconcentration of 6 mM MgCl₂, but it was found herein that higher yieldis obtained when increasing the concentration to 9 mM or beyond.Finally, the average length of the preamplified cDNA increased with 370nts when administering dNTPs prior to the RNA denaturation rather thanin the RT master mix (p=7.8×10⁻⁹, Student's t-test; FIG. 1c ).

It was further demonstrated that these improvements obtained withpurified RNA extended to cDNA reactions performed directly in lysates ofindividual human and mouse cells. To this end, single-cell cDNAlibraries were generated from a total of 262 individual human or mousecells (159 HEK293T, 34 DG-75, 30 C2C12 and 39 MEF cells) spanningdifferent cell sizes and total RNA contents (Table 3). Analyses of thesingle-cell cDNA libraries demonstrated higher cDNA yields both with theuse of the LNA-containing TSO (3-fold increase, p<0.001, Student'st-test; FIG. 1d ) and with betaine together with high Mg²⁺concentrations (4-fold increase, p=3.7×10⁻⁶, Student's t-test; FIG. 1e).

The sensitivity and accuracy of single-cell methods are limited by theefficiency of each sample-processing step. The SMARTer® protocol usesbead purification to remove unincorporated adaptors from the firststrand cDNA reaction before the preamplification with Advantage 2Polymerase (Adv2). However, performing bead purification in smallvolumes poses a significant recovery challenge for liquid handlingautomation. It was determined herein that KAPA HiFi Hot Start (KAPA) DNAPolymerase efficiently amplified first-strand cDNA directly afterreverse transcription, with no need for prior bead purification.Libraries preamplified without bead purification had no reduction inyield, but the average cDNA length increased with 450 nts (p=2.6×10⁻¹²,Student's t-test; FIG. 10 demonstrating that KAPA preamplificationimproves cDNA generation and offers a viable approach for Smart-Seqautomation.

To demonstrate the significance of the improved cDNA generation ondownstream applications, its impact on single-cell transcriptomeprofiling was assessed. To this end, single HEK293T cell librariesgenerated both according to the commercial SMARTer (n=4) and usingvariations of the present protocol were sequenced (Smart-Seq2, n=35)(Table 4).

The improved conversion of RNA to cDNA should improve gene expressionprofiling as more original RNA molecules are accessible for sequencing.Indeed, both a significant increase in the ability to detect geneexpression (FIG. 2a ) and lowered technical variation for low and mediumabundance transcripts were observed (FIG. 2b and FIG. 4). The improvedsensitivity of the optimized protocol led to the average detection of2,372 more genes in each cell (p<0.05 Student's t-test; FIG. 2c ). Allthese improvements were independently validated using an alternativeRNA-Seq alignment and analyses strategy (FIG. 5). Moreover, both bettersensitivity and lower variability in single-cell transcriptome datagenerated with Smart-Seq2 than for data available for Quartz-Seq wereobtained (FIG. 6). Although the sequenced libraries had similar mappingscharacteristics as SMARTer libraries, a 7% increase in unmapped readswas noted (FIG. 7).

Several preamplification enzymes have lower GC bias than the Advantage2(Adv2) that is used with SMARTer®, indicating that single-cell profilingcould also improve with cDNA preamplifications using KAPA. Indeed, thesingle-cell libraries preamplified with KAPA detected more genes athigher GC levels (FIG. 2d and FIG. 8) and improved sensitivity andaccuracy (FIG. 9). When compared with the low coverage of 5′ regions insingle-cell data generated through 3′ end polyA-tailing of cDNA,single-cell RNA-Seq libraries that had been preamplified with KAPA hadsignificantly better coverage across the full length of transcripts(p<10⁻⁵, 1.6×10⁻³ for 5′ and 3′ ends respectively, Student's t-test), asthey approached the expected fraction of reads at the 5′ and 3′ ends(FIG. 2e and FIGS. 10-11). Importantly, global gene expression profilesfrom cells preamplified with KAPA and Adv2 separated on the firstprincipal component (FIG. 2f ), demonstrating that preamplification biashad significant impact on absolute expression levels. Regions withartificially large number of reads aligned (i.e. peak) appearingsystematically in Smart-Seq irrespectively of preamplification enzymethat necessitated filtering were sequenced (FIG. 12). Together, the datashow that preamplification using KAPA improved GC tolerance and readcoverage across transcripts.

To determine the extent of technical variability in the single-celltranscriptome profiling with Smart-Seq2, sequencing libraries weregenerated from dilution series of HEK293T cells (100, 50 and 10 cells)and total RNA (1 ng, 100 pg, 10 pg). Technical losses and variationswere small when analyzing 10 cells or more, but considerable variabilityexists at single-cell levels, as previously observed. It is informativeto contrast the technical variability with the biological variabilitypresent in cells of the same or different cell type origin (FIG. 13A-D).To this end, additional single-cell transcriptomes were sequenced fromDG-75 (n=7), C2C12 (n=6) and MEF (n=7) cells. Analyzing the biologicalvariability between and within cell populations revealed that biologicalvariability associated with cell type specific expression exceededtechnical variability at around 50 RPKM, but the exact threshold willdepend on the RNA content present in the cell types studied (FIG. 13E).

This invention provides a new protocol that improves sensitivity,accuracy and coverage across transcripts and is more amenable toautomation. Moreover, the new protocol costs less than 12% of thecurrent cost per reaction and only 3% when using in-house produced Tn5(FIG. 14).

Although these results were reached in the context of Smart-Seqsingle-cell gene expression analyses, these modifications are applicableother single-cell methods that rely on template switching, includingthose carried out on microfluidic chips (e.g. Fluidigm C1) or insideemulsion droplets.

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thespirit and script of the invention, and all such variations are intendedto be included within the scope of the following claims.

All references cited herein are incorporated herein by reference intheir entireties.

TABLE S1 Tables of cDNA library yield and length starting with purifiedtotal RNA Worksheet A lists all 457 cDNA libraries generated from mousebrain total RNA. The general protocol followed for each sample isindicated in the “general protocol” column, with specific information onthe template switching oligonucleotide, RT enzyme, PCR enzyme, MgCl2concentration, betaine, bead purification and dNTPs administrationtiming detailed in separate columns. Worksheet B contains a list ofdirect comparisons of variables that effect cDNA library yield andaverage length using replicate groups that have identical reactionparameters except for the experimental variable evaluated. amount dil.TSO (ul dNTPs Bio- avg of 10 uM purifica- PCR added amount PCR ul ana-additional conc size in 10 ul RT MgCl2 RT betaine tion PCR rxn vol inthe other Entry (ng) cycles elution lyzer description (pg/ul) (bp) TSORT rxn) enzyme (mM) protocol (M) after RT enzyme (ul) beginning?additives 1 1 15 10 1:5 SMARTer kit 318 1669 SMARTer 1 SMARTscribe 6 90′@ — yes Advantage 50 — — Oligo IIA 42° C. 2 1 15 10 1:5 SMARTer kit 2631857 SMARTer 1 SMARTscribe 6 90′ @ — yes Advantage 50 — — but Oligo IIA42° C. replacing ISPCR primers 3 1 15 10 1:5 SMARTer kit 172 1784SMARTer 1 SMARTscribe 6 90′ @ — yes Advantage 50 — — but Oligo IIA 42°C. replacing oligo dT 4 SMARTer kit FAILED SMARTer 1 SMARTscribe 6 90′ @— yes Advantage 50 — 3 mM but Oligo IIA 42° C. MnCl2 replacing FSB 5 115 10 1:5 modified 393 1683 SMARTer 1 SSRTII 6 90′ @ — yes Advantage 50— — SMARTer kit Oligo IIA 42° C. 6 1 15 NA NA modified 121 1780 rG5 1SMARTscribe 6 90′ @ — yes Advantage 50 — — SMARTer kit 42° C. 7 1 15 NANA modified 1038 1346 rGrGrG 1 SMARTscribe 6 90′ @ — yes Advantage 50 —— SMARTer kit 42° C. 8 1 15 NA NA modified 90 1652 rGrGrGp 1 SMARTscribe6 90′ @ — yes Advantage 50 — — SMARTer kit 42° C. 9 1 15 NA NA modified84 1608 ISO 1 SMARTscribe 6 90′ @ — yes Advantage 50 — — SMARTer kit 42°C. 10 1 15 NA NA SMARTer kit 3628 1838 SMARTer 1 SMARTscribe 6 90′ @ —yes Advantage 50 — — Oligo IIA 42° C. 11 1 15 NA NA modified 650 2108rG5 1 SMARTscribe 6 90′ @ — yes Advantage 50 — — SMARTer kit 42° C. 12 115 NA NA modified 6423 1852 rGrGrG 1 SMARTscribe 6 90′ @ — yes Advantage50 — — SMARTer kit 42° C. 13 1 15 NA NA modified 666 2187 rGrGrGp 1SMARTscribe 6 90′ @ — yes Advantage 50 — — SMARTer kit 42° C. 14 1 15 NANA modified 525 2181 ISO 1 SMARTscribe 6 90′ @ — yes Advantage 50 — —SMARTer kit 42° C. 15 1 15 NA NA SMARTer kit 3366 1782 SMARTer 1SMARTscribe 6 90′ @ — yes Advantage 50 — — but Oligo IIA 42° C.replacing ISPCR primers 16 1 15 NA NA modified 435 1970 ds oligos 1SMARTscribe 6 90′ @ — yes Advantage 50 — — SMARTer kit 42° C. 17 1 15 NANA SMARTer kit 1143 1445 SMARTer 1 SMARTscribe 6 90′ @ — yes Advantage50 — — Oligo IIA 42° C. 18 1 15 NA NA SMARTer kit 1504 1519 SMARTer 1SMARTscribe 6 90′ @ — yes Advantage 50 — — but Oligo IIA 42° C.replacing ISPCR primers 19 1 15 NA NA SMARTer kit 1984 1728 SMARTer 1SMARTscribe 6 90′ @ — yes Advantage 50 — — but Oligo IIA 42° C.replacing ISPCR primers 20 1 15 NA NA modified 1346 1543 SMARTer 1SSRTII 6 90′ @ — yes Advantage 50 — — SMARTer kit Oligo IIA 42° C. 21 115 NA NA modified 1191 1683 SMARTer 1 SSRTII 6 90′ @ — yes Advantage 50— — SMARTer kit Oligo IIA 42° C. 22 1 15 NA NA SMARTer kit 103 1420SMARTer 1 SMARTscribe 3 90′ @ — yes Advantage 50 — — but using Oligo IIA42° C. Superscript II FSB (3 mM MgCl2) 23 1 15 NA NA SMARTer kit 1921609 SMARTer 1 SMARTscribe 3 90′ @ — yes Advantage 50 — — but usingOligo IIA 42° C. Superscript II FSB (3 mM MgCl2 24 SMARTer kit FAILEDSMARTer 1 SMARTscribe — 90′ @ — yes Advantage 50 — 6 mM but using OligoIIA 42° C. MnCl2 STRT buffer 25 1 15 NA NA SMARTer kit 1573 2378 SMARTer1 SMARTscribe 6 90′ @ — yes Advantage 50 — — Oligo IIA 42° C. 26 1 15 NANA modified 246 2024 rG5 1 SMARTscribe 3 90′ @ — yes Advantage 50 — —SMARTer kit 42° C. 27 1 15 NA NA modified 1393 1945 rGrGrG 1 SMARTscribe3 90′ @ — yes Advantage 50 — — SMARTer kit 42° C. 28 1 15 NA NA modified259 2180 rGrGrGp 1 SMARTscribe 3 90′ @ — yes Advantage 50 — — SMARTerkit 42° C. 29 1 15 NA NA modified 776 1958 SMARTer 1 SMARTscribe 6 90′ @1 yes Advantage 50 — — SMARTer kit Oligo IIA 42° C. 30 1 15 NA NAmodified 731 1808 SMARTer 1 SMARTscribe 6 90′ @ 2 yes Advantage 50 — —SMARTer kit Oligo IIA 42° C. 31 1 15 NA NA modified 1936 1965 SMARTer 1SMARTscribe 6 90′ @ 1 yes Advantage 50 — — SMARTer kit Oligo IIA 42° C.32 modified FAILED SMARTer 1 SMARTscribe 6 90′ @ 1 yes Advantage 50 — 3mM SMARTer kit Oligo IIA 42° C. MnCl2 33 modified FAILED SMARTer 1SMARTscribe 6 90′ @ — yes Advantage 50 — 3 mM SMARTer kit Oligo IIA 42°C. MnCl2, 5% DMSO 34 1 15 NA NA SMARTer kit 580 2634 SMARTer 1SMARTscribe 6 90′ @ — yes Advantage 50 — — Oligo IIA 42° C. 35 1 15 NANA modified 166 2208 SMARTer 1 SMARTscribe 3 90′ @ 1 yes Advantage 50 —— SMARTer kit Oligo IIA 42° C. 36 1 15 NA NA modified 204 2016 SMARTer 1SMARTscribe 3 90′ @ 1 yes Advantage 50 — — SMARTer kit Oligo IIA 42° C.37 1 15 NA NA modified 547 2171 SMARTer 1 SMARTscribe 6 90′ @ 1 yesAdvantage 50 — — SMARTer kit Oligo IIA 42° C. 38 1 15 NA NA modified 6402028 SMARTer 1 SMARTscribe 6 90′ @ 1 yes Advantage 50 — — SMARTer kitOligo IIA 42° C. 39 1 15 NA NA modified 145 1938 rGrGrG 1 SMARTscribe 390′ @ 1 yes Advantage 50 — — SMARTer kit 42° C. 40 1 15 NA NA modified224 1841 rGrGrG 1 SMARTscribe 3 90′ @ 1 yes Advantage 50 — — SMARTer kit42° C. 41 1 15 NA NA modified 496 1863 rGrGrG 1 SMARTscribe 6 90′ @ 1yes Advantage 50 — — SMARTer kit 42° C. 42 1 15 NA NA modified 493 1911rGrGrG 1 SMARTscribe 6 90′ @ 1 yes Advantage 50 — — SMARTer kit 42° C.43 1 15 20 — in house prot 8431 2226 rGrGrG 1 SSRTII 6 90′ @ 1 yesAdvantage 50 — — 42° C. 44 1 15 20 — in house prot 8248 2235 rGrGrG 1SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 45 1 15 20 — in house prot7672 2317 rGrGrG 1 SSRTII 6 90′ @ 1.5 yes Advantage 50 — — 42° C. 46 115 20 — in house prot 6562 2248 rGrGrG 1 SSRTII 6 90′ @ 1.5 yesAdvantage 50 — — 42° C. 47 1 15 20 — SMARTer kit 8156 2298 SMARTer 1SMARTscribe 6 90′ @ — yes Advantage 50 — — Oligo IIA 42° C. 48 1 15 20 —in house prot 1744 2592 rGrGrGp 1 SSRTII 6 90′ @ 1 yes Advantage 50 — —42° C. 49 1 15 20 — in house prot 1820 2585 rGrGrGp 1 SSRTII 6 90′ @ 1yes Advantage 50 — — 42° C. 50 1 15 20 — in house prot 1591 2541 rGrGrGp1 SSRTII 6 90′ @ 1.5 yes Advantage 50 — — 42° C. 51 1 15 20 — in houseprot 1398 2616 rGrGrGp 1 SSRTII 6 90′ @ 1.5 yes Advantage 50 — — 42° C.52 1 15 20 — in house prot 2265 2110 dGCGGG 1 SSRTII 6 90′ @ 1 yesAdvantage 50 — — 42° C. 53 1 15 20 — in house prot 2568 2125 dGCGGG 1SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 54 1 15 20 — in house prot2263 2079 dGCGGG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 55 1 1520 — in house prot 691 2290 dGCGGGp 1 SSRTII 6 90′ @ 1 yes Advantage 50— — 42° C. 56 1 15 20 — in house prot 757 2344 dGCGGGp 1 SSRTII 6 90′ @1 yes Advantage 50 — — 42° C. 57 1 15 20 — in house prot 690 2294dGCGGGp 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 58 1 15 20 —SMARTer kit 6525 2295 SMARTer 1 SMARTscribe 6 90′ @ — yes Advantage 50 —— Oligo IIA 42° C. 59 1 15 20 — SMARTer kit 6806 2270 SMARTer 1SMARTscribe 6 90′ @ — yes Advantage 50 — — Oligo IIA 42° C. 60 1 15 20 —in house prot, 5922 1861 rGrGrG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — —processed 42° C. immediately after adding lysis buffer (LB) 61 1 15 20 —in house prot, 5419 1892 rGrGrG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — —processed 42° C. immediately after adding lysis buffer (LB) 62 1 15 20 —in house prot, 5664 1806 rGrGrG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — —processed 42° C. immediately after adding lysis buffer (LB), 60′ afteradding lysis buffer (LB), stored at RT 63 1 15 20 — in house prot, 55541800 rGrGrG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — processed 42° C.immediately after adding lysis buffer (LB), 45′ after adding lysisbuffer (LB), stored at RT 64 1 15 20 — in house prot, 5552 1772 rGrGrG 1SSRTII 6 90′ @ 1 yes Advantage 50 — — processed 42° C. immediately afteradding lysis buffer (LB), 30′ after adding lysis buffer (LB), stored atRT 65 1 15 20 — in house prot, 6335 1824 rGrGrG 1 SSRTII 6 90′ @ 1 yesAdvantage 50 — — processed 42° C. immediately after adding lysis buffer(LB), 10′ after adding lysis buffer (LB), stored at RT 66 1 15 20 — inhouse prot, 4904 1700 rGrGrG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — —processed 42° C. immediately after adding lysis buffer (LB), 60′ afteradding lysis buffer (LB), stored in the fridge 67 1 15 20 — in houseprot, 3938 1544 rGrGrG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — processed42° C. immediately after adding lysis buffer (LB), 45′ after addinglysis buffer (LB), stored in the fridge 68 1 15 20 — in house prot, 37931585 rGrGrG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — processed 42° C.immediately after adding lysis buffer (LB), 30′ after adding lysisbuffer (LB), stored in the fridge 69 1 15 20 — in house prot, 5312 1824rGrGrG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — processed 42° C.immediately after adding lysis buffer (LB), 10′ after adding lysisbuffer (LB), stored in the fridge 70 1 15 30 — in house prot, 1024 1547rGrGrG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — 3 mM RT for 42° C. MnCl2 1h @42° C., then added 3 mM MnCl2 and incubated for 15′ 71 1 15 30 — inhouse prot, 849 1412 rGrGrG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — 3 mMRT for 42° C. MnCl2 1 h @42° C., then added 3 mM MnCl2 and incubated for15′ 72 1 15 30 — in house prot, 558 1227 rGrGrG 1 SSRTII 6 90′ @ 1 yesAdvantage 50 — 6 mM RT for 42° C. MnCl2 1 h @42° C., then added 6 mMMnCl2 and incubated for 15′ 73 1 15 30 — in house prot, 572 1199 rGrGrG1 SSRTII 6 90′ @ 1 yes Advantage 50 — 6 mM RT for 42° C. MnCl2 1 h @42°C., then added 6 mM MnCl2 and incubated for 15′ 74 1 15 30 — in houseprot 2105 2051 rGrGrG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 751 15 30 — in house prot 1664 1809 rGrGrG 1 SSRTII 6 90′ @ 1 yesAdvantage 50 — — 42° C. 76 100 10 15 1:5 in house prot 3196 1946 2OMe 1SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 77 100 10 15 1:5 in houseprot 8894 1767 rGrG + G 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C.78 100 10 15 1:5 in house prot 1939 2054 ddC 1 SSRTII 6 90′ @ 1 yesAdvantage 50 — — 42° C. 79 100 10 15 1:5 in house prot 1942 1286 rGrGrG1 SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 80 10 12 15 1:5 in houseprot 1635 1891 2OMe 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 81 1012 15 1:5 in house prot 1230 1705 2OMe 1 SSRTII 6 90′ @ 1 yes Advantage50 — — 42° C. 82 10 12 15 1:5 in house prot 4700 1602 rGrG + G 1 SSRTII6 90′ @ 1 yes Advantage 50 — — 42° C. 83 10 12 15 1:5 in house prot 40511717 rGrG + G 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 84 10 12 151:5 in house prot 733 1904 ddC 1 SSRTII 6 90′ @ 1 yes Advantage 50 — —42° C. 85 10 12 15 1:5 in house prot 637 1897 ddC 1 SSRTII 6 90′ @ 1 yesAdvantage 50 — — 42° C. 86 10 12 15 1:5 in house prot 1104 1556 rGrGrG 1SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 87 1 15 15 1:5 in houseprot 923 1734 2OMe 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 88 115 15 1:5 in house prot 842 1620 2OMe 1 SSRTII 6 90′ @ 1 yes Advantage50 — — 42° C. 89 1 15 15 1:5 in house prot 3387 1426 rGrG + G 1 SSRTII 690′ @ 1 yes Advantage 50 — — 42° C. 90 1 15 15 1:5 in house prot 36091473 rGrG + G 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 91 1 15 151:5 in house prot 401 1463 ddC 1 SSRTII 6 90′ @ 1 yes Advantage 50 — —42° C. 92 1 15 15 1:5 in house prot 470 1661 ddC 1 SSRTII 6 90′ @ 1 yesAdvantage 50 — — 42° C. 93 1 15 15 1:5 in house prot 1550 1341 rGrGrG 1SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 94 1 15 15 1:5 in houseprot 1267 1483 rGrGrG 1 SSRTII 6 90′ @ 1 yes Advantage 50 — — 42° C. 951 15 15 1:5 in house prot 2176 1438 rGrG + G 1 SSRTII 6 90′ @ 1 yesAdvantage 50 — — 42° C. 96 1 15 15 1:5 in house prot 1469 1705 rGrGrG 1SSRTII 9 90′ @ 1 yes Advantage 50 — — 42° C. 97 1 15 15 1:5 in houseprot 2383 1596 rGrG + G 1 SSRTII 9 90′ @ 1 yes Advantage 50 — — 42° C.98 1 15 15 1:5 in house prot 1431 1807 rGrGrG 1 SSRTII 12 90′ @ 1 yesAdvantage 50 — — 42° C. 99 1 15 15 1:5 in house prot 3630 1623 rGrG + G1 SSRTII 12 90′ @ 1 yes Advantage 50 — — 42° C. 100 1 15 15 1:5 in houseprot 1277 1610 rGrGrG 1 SSRTII 15 90′ @ 1 yes Advantage 50 — — 42° C.101 1 15 15 1:5 in house prot 1884 1462 rGrG + G 1 SSRTII 15 90′ @ 1 yesAdvantage 50 — — 42° C. 102 1 15 15 1:5 in house prot 1123 1652 SMARTer1 SSRTII 6 90′ @ 1 yes Advantage 50 — — Oligo IIA 42° C. 103 1 15 15 1:5in house prot 935 1593 SMARTer 1 SSRTII 15 90′ @ 1 yes Advantage 50 — —Oligo IIA 42° C. 104 1 15 15 1:5 in house prot, 2156 1767 rGrG + G 1SSRTII 12 90′ @ 1 yes KAPA 50 — — KAPA HiFi 42° C. HiFi HS after washingbeads 1 105 1 15 15 1:5 in house prot, 352 2151 rGrGrG 1 SSRTII 12 90′ @1 yes KAPA 50 — — KAPA HiFi 42° C. HiFi HS after washing beads 1 106 115 15 1:5 in house prot, 1729 1611 rGrG + G 1 SSRTII 12 90′ @ 1 yes KAPA50 — — KAPA HiFi 42° C. HiFi HS after washing beads 2 107 1 15 15 1:5 inhouse prot, 203 2073 rGrGrG 1 SSRTII 12 90′ @ 1 yes KAPA 50 — — KAPAHiFi 42° C. HiFi HS after washing beads 2 108 1 15 15 1:5 in house prot,1108 1480 rGrG + G 1 SSRTII 12 90′ @ 1 yes KAPA 50 — — KAPA HiFi 42° C.HiFi HS after washing beads 1 + elution 109 1 15 15 1:5 in house prot,301 1903 rGrGrG 1 SSRTII 12 90′ @ 1 yes KAPA 50 — — KAPA HiFi 42° C.HiFi HS after washing beads 1 + elution 110 1 15 15 1:5 in house prot,1337 1522 rGrG + G 1 SSRTII 12 90′ @ 1 yes KAPA 50 — — KAPA HiFi 42° C.HiFi HS after washing beads 2 + elution 112 1 15 15 1:5 in house prot,383 1935 rGrGrG 1 SSRTII 12 90′ @ 1 yes KAPA 50 — — KAPA HiFi 42° C.HiFi HS after washing beads 2 + elution 113 1 15 15 1:5 in house prot2897 1737 rGrG + G 1 SSRTII 12 90′ @ 1 yes Advantage 50 — — 42° C. 114 115 15 1:5 in house prot 1853 1706 rGrGrG 1 SSRTII 12 90′ @ 1 yesAdvantage 50 — — 42° C. 115 1 15 15 1:5 in house prot 1492 1563 rGrG + G1 Revertaid 4 90′ @ 1 yes Advantage 50 — — H− 42° C. 116 1 15 15 1:5 inhouse prot 1236 1472 rGrG + G 1 Revertaid 4 90′ @ 1 yes Advantage 50 — —H− 42° C. 117 1 15 15 1:5 in house prot 1843 1450 rGrG + G 1 Revertaid 690′ @ 1 yes Advantage 50 — — H− 42° C. 118 1 15 15 1:5 in house prot1465 1346 rGrG + G 1 Revertaid 6 90′ @ 1 yes Advantage 50 — — H− 42° C.119 1 15 15 1:5 in house prot 2996 1597 rGrG + G 1 Revertaid 9 90′ @ 1yes Advantage 50 — — H− 42° C. 120 1 15 15 1:5 in house prot 2654 1530rGrG + G 1 Revertaid 9 90′ @ 1 yes Advantage 50 — — H− 42° C. 121 1 1515 1:5 in house prot 2159 1487 rGrG + G 1 Revertaid 12 90′ @ 1 yesAdvantage 50 — — H− 42° C. 122 1 15 15 1:5 in house prot 1890 1412rGrG + G 1 Revertaid 12 90′ @ 1 yes Advantage 50 — — H− 42° C. 123 1 1515 1:5 in house prot 1504 1246 rGrG + G 1 Revertaid 15 90′ @ 1 yesAdvantage 50 — — H− 42° C. 124 1 15 15 1:5 in house prot 1986 1474rGrG + G 1 Revertaid 15 90′ @ 1 yes Advantage 50 — — H− 42° C. 125 1 1515 1:5 in house prot 1109 1691 rGrG + G 1 SSRTII — 60′ @42° — yesAdvantage 50 — 0.3M C., then trehalose 90′ @60° C. 126 1 15 15 1:5 inhouse prot 1090 1752 rGrG + G 1 SSRTII — 60′ @42° — yes Advantage 50 —0.3M C., then trehalose 90′ @60° C. 127 1 15 15 1:5 in house prot 8631565 rGrG + G 1 SSRTII — 60′ @42° — yes Advantage 50 — 0.6M C., thentrehalose 90′ @60° C. 128 1 15 15 1:5 in house prot 896 1652 rGrG + G 1SSRTII — 60′ @42° — yes Advantage 50 — 0.6M C., then trehalose 90′ @60°C. 129 1 15 15 1:5 in house prot 1078 1655 rGrG + G 1 SSRTII — 60′ @42°1 yes Advantage 50 — 0.3M C., then trehalose 90′ @60° C. 130 1 15 15 1:5in house prot 562 1517 rGrG + G 1 SSRTII — 60′ @42° 1 yes Advantage 50 —0.3M C., then trehalose 90′ @60° C. 131 1 15 15 1:5 in house prot 9981594 rGrG + G 1 SSRTII — 60′ @42° 0.6 yes Advantage 50 — 0.3M C., thentrehalose 90′ @60° C. 132 1 15 15 1:5 in house prot 925 1545 rGrG + G 1SSRTII — 60′ @42° 0.6 yes Advantage 50 — 0.3M C., then trehalose 90′@60° C. 133 1 15 15 1:5 in house prot 1155 1618 rGrG + G 1 SSRTII 3 60′@42° 1 yes Advantage 50 — 0.3M C., then trehalose 90′ @60° C. 134 1 1515 1:5 in house prot 603 1433 rGrG + G 1 SSRTII 3 60′ @42° 1 yesAdvantage 50 — 0.3M C., then trehalose 90′ @60° C. 135 1 15 15 1:5 inhouse prot 1561 1716 rGrG + G 1 SSRTII 3 60′ @42° 0.6 yes Advantage 50 —0.3M C., then trehalose 90′ @60° C. 136 1 15 15 1:5 in house prot 4451575 rGrG + G 1 SSRTII — 60′ @50° — yes Advantage 50 — 0.3M C., thentrehalose 90′ @42° C. 137 1 15 15 1:5 in house prot 1466 1698 rGrG + G 1SSRTII — 90′ @42° — yes Advantage 50 — 0.3M C., trehalose 30′ @60° C.,then 30′ @42° C. 138 1 15 15 1:5 in house prot 703 1580 rGrG + G 1SSRTII — 60′ @50° — yes Advantage 50 — 0.6M C., then trehalose 90′ @42°C. 139 1 15 15 1:5 in house prot 1397 1740 rGrG + G 1 SSRTII — 90′ @42°— yes Advantage 50 — 0.6M C., trehalose 30′ @60° C., then 30′ @42° C.140 1 15 15 1:5 in house prot 355 1425 rGrG + G 1 SSRTII — 60′ @50° 0.6yes Advantage 50 — 0.6M C., then trehalose 90′ @42° C. 141 1 15 15 1:5in house prot 1654 1598 rGrG + G 1 SSRTII — 90′ @42° 0.6 yes Advantage50 — 0.6M C., trehalose 30′ @60° C., then 30′ @42° C. 142 1 15 15 1:5 inhouse prot 1470 1480 rGrG + G 1 SSRTII 12 60′ @50° 0.6 yes Advantage 50— 0.6M C., then trehalose 90′ @42° C. 143 1 15 15 1:5 in house prot 13891480 rGrG + G 1 SSRTII 12 90′ @42° 0.6 yes Advantage 50 — 0.6M C.,trehalose 30′ @60° C., then 30′ @42° C. 144 1 15 15 1:5 in house prot3959 1641 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then10X (2′ @50° C.- 2′ @42° C.) 145 1 15 15 1:5 in house prot 3816 1732rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then 10X (2′@60° C.- 2′ @42° C. 146 1 15 15 1:5 in house prot 3179 1769 rGrG + G 1SSRTII 12 90′ @42° 0.5 yes Advantage 50 — 0.3M C., then 10X trehalose(2′ @50° C.- 2′ @42° C. 147 1 15 15 1:5 in house prot 3271 1828 rGrG + G1 SSRTII 12 90′ @42° 0.5 yes Advantage 50 — 0.3M C., then 10X trehalose(2′ @60° C.- 2′ @42° C.) 148 1 15 15 1:5 in house prot 4081 1706 rGrG +G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then 5x (2′ @50° C.-2′ @42° C.) 149 1 15 15 1:5 in house prot 3858 1771 rGrG + G 1 SSRTII 1290′ @42° 1 yes Advantage 50 — — C., then 5x (2′ @50° C.- 2′ @42° C.) 1501 15 15 1:5 in house prot 3711 1781 rGrG + G 1 SSRTII 12 90′ @42° 1 yesAdvantage 50 — — C., then 5x (2′ @50° C.- 2′ @42° C.) 151 1 15 15 1:5 inhouse prot 4015 1773 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 —— C., then 10x (2′ @50° C.- 2′ @42° C.) 152 1 15 15 1:5 in house prot3671 1753 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then10x (2′ @50° C.- 2′ @42° C.) 153 1 15 15 1:5 in house prot 3498 1708rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then 10x (2′@50° C.- 2′ @42° C.) 154 1 15 15 1:5 in house prot 3804 1610 rGrG + G 1SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then 15x (2′ @50° C.- 2′@42° C.) 155 1 15 15 1:5 in house prot 3613 1679 rGrG + G 1 SSRTII 1290′ @42° 1 yes Advantage 50 — — C., then 15x (2′ @50° C.- 2′ @42° C.)156 1 15 15 1:5 in house prot 4595 1630 rGrG + G 1 SSRTII 12 90′ @42° 1yes Advantage 50 — — C., then 15x (2′ @50° C.- 2′ @42° C.) 157 1 15 151:5 in house prot 3457 1525 rGrG + G 1 SSRTII 12 90′ @42° 1 yesAdvantage 50 — — C., then 20x (2′ @50° C.- 2′ @42° C.) 158 1 15 15 1:5in house prot 2869 1409 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50— — C., then 20x (2′ @50° C.- 2′ @42° C.) 159 1 15 15 1:5 in house prot1529 1629 3rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then10x (2′ @50° C.- 2′ @42° C.) 160 1 15 15 1:5 in house prot 1901 17283rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then 10x (2′@50° C.- 2′ @42° C.) 161 1 15 15 1:5 in house prot 1785 1717 3rGrG + G 1SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then 10x (2′ @50° C.- 2′@42° C.) 162 1 15 15 1:5 in house prot 1086 1928 2rGrG + G 1 SSRTII 1290′ @42° 1 yes Advantage 50 — — C., then 10x (2′ @50° C.- 2′ @42° C.)163 1 15 15 1:5 in house prot 1128 1846 2rGrG + G 1 SSRTII 12 90′ @42° 1yes Advantage 50 — — C., then 10x (2′ @50° C.- 2′ @42° C.) 164 1 15 151:5 in house prot 596 1892 phosphate 1 SSRTII 12 90′ @42° 1 yesAdvantage 50 — — C., then 10x (2′ @50° C.- 2′ @42° C.) 165 1 15 15 1:5in house prot 579 1922 phosphate 1 SSRTII 12 90′ @42° 1 yes Advantage 50— — C., then 10x (2′ @50° C.- 2′ @42° C.) 166 1 15 15 1:5 in house prot546 1830 C6 amino 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then10x (2′ @50° C.- 2′ @42° C.) 167 1 15 15 1:5 in house prot 419 1664 C6amino 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then 10x (2′ @50°C.- 2′ @42° C.) 168 1 15 15 1:5 in house prot 3076 1567 rGrG + G 1SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then 10x (2′ @50° C.- 2′@42° C.) 169 1 15 15 1:5 in house prot 2889 1465 rGrG + G 1 SSRTII 1290′ @42° 1 yes Advantage 50 — — C., then 10x (2′ @50° C.- 2′ @42° C.)170 1 15 15 1:5 in house prot 3653 1735 rGrG + G 1 SSRTII 12 90′ @42° 1yes Advantage 50 — — C., then 10x (2′ @50° C.- 2′ @42° C.) 171 1 15 151:5 in house prot 2152 1455 rGrG + G 1 SSRTII 12 90′ @42° 1 yesAdvantage 50 — — C., then 10x (2′ @50° C.- 2′ @42° C.) 172 1 15 15 1:5in house prot 1454 1084 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50— 0.816M C., then 10x 1,2 (2′ @50° C.- propandiol 2′ @42° C.) 173 1 1515 1:5 in house prot 1331 1106 rGrG + G 1 SSRTII 12 90′ @42° 1 yesAdvantage 50 — 0.816M C., then 10x 1,2 (2′ @50° C.- propandiol 2′ @42°C.) 174 1 15 15 1:5 in house prot 1373 1089 rGrG + G 1 SSRTII 12 90′@42° 1 yes Advantage 50 — 0.816M C., then 10x 1,2 (2′ @50° C.-propandiol 2′ @42° C.) 175 1 15 15 1:5 in house prot 1904 1453 rGrG + G1 SSRTII 12 90′ @42° 1 yes Advantage 50 — 1.075M C., then 10x ethylene(2′ @50° C.- glycol 2′ @42° C.) 176 1 15 15 1:5 in house prot 2855 1390rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — 1.075M C., then 10xethylene (2′ @50° C.- glycol 2′ @42° C.) 177 1 15 15 1:5 in house prot2571 1670 rGrG + G 1 Maxima 4 90′ @50° 1 yes Advantage 50 — — H− C. 1781 15 15 1:5 in house prot 2549 1722 rGrG + G 1 Maxima 4 90′ @50° 1 yesAdvantage 50 — — H− C. 179 1 15 15 1:5 in house prot 288 1599 rGrG + G 1Revertaid 4 90′ @50° 1 yes Advantage 50 — — Premium C. 180 1 15 15 1:5in house prot 129 1608 rGrG + G 1 Revertaid 4 90′ @50° 1 yes Advantage50 — — Premium C. 181 in house prot FAILED rGrG + G 1 Maxima 12 90′ @50°1 yes Advantage 50 — — H− C. 182 in house prot FAILED rGrG + G 1 Maxima12 90′ @50° 1 yes Advantage 50 — — H− C. 183 1 15 15 1:5 in house prot2633 1341 rGrG + G 1 Revertaid 12 90′ @50° 1 yes Advantage 50 — —Premium C. 184 1 15 15 1:5 in house prot 2662 1325 rGrG + G 1 Revertaid12 90′ @50° 1 yes Advantage 50 — — Premium C. 185 1 15 15 1:5 in houseprot 2814 1674 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — — C.,then 10x (2′ @50° C.- 2′ @42° C.) 186 in house prot FAILED rGrG + G 1SSRTII 12 90′ @42° 1 yes Long PCR 50 — — C., then 10x Enzyme (2′ @50°C.- mix 2′ @42° C.) 187 1 15 15 1:5 in house prot, 3155 1764 rGrG + G 1SSRTII 12 90′ @42° 1 — Phusion 100 — — PCR w/o C., then 10x HS purif in(2′ @50° C.- 100 ul 2′ @42° C.) 188 1 15 15 1:5 in house prot, 2740 1793rGrG + G 1 SSRTII 12 90′ @42° 1 — Phusion 100 — — PCR w/o C., then 10xHS purif in (2′ @50° C.- 100 ul 2′ @42° C.) 189 1 15 15 1:5 in houseprot, 1857 1739 rGrG + G 1 SSRTII 12 90′ @42° 1 — Q5 NEB 100 — — PCR w/oC., then 10x purif in (2′ @50° C.- 100 ul 2′ @42° C.) 190 1 15 15 1:5 inhouse prot, 1697 1697 rGrG + G 1 SSRTII 12 90′ @42° 1 — Q5 NEB 100 — —PCR w/o C., then 10x purif in (2′ @50° C.- 100 ul 2′ @42° C.) 191 1 1515 1:5 in house prot, 3278 1639 rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA100 — — PCR w/o C., then 10x HiFi HS purif in (2′ @50° C.- 100 ul 2′@42° C.) 192 1 15 15 1:5 in house prot, 3719 1590 rGrG + G 1 SSRTII 1290′ @42° 1 — KAPA 100 — — PCR w/o C., then 10x HiFi HS purif in (2′ @50°C.- 100 ul 2′ @42° C.) 193 1 15 15 1:5 in house prot, 3816 1630 rGrG + G1 SSRTII 12 90′ @42° 1 — Advantage 50 — — PCR w/o C., then 10x purif in(2′ @50° C.- 50 ul 2′ @42° C.) 194 1 15 15 1:5 in house prot, 2462 1653rGrG + G 1 SSRTII 12 90′ @42° 1 — Advantage 50 — — PCR w/o C., then 10xpurif in (2′ @50° C.- 50 ul 2′ @42° C.) 195 1 15 15 1:5 in house prot,2848 1588 rGrG + G 1 SSRTII 12 90′ @42° 1 — Advantage 50 — — PCR w/o C.,then 10x purif in (2′ @50° C.- 50 ul 2′ @42° C.) 196 1 15 15 1:5 inhouse prot, 2012 1669 rGrG + G 1 SSRTII 12 90′ @42° 1 — Phusion 50 — —PCR w/o C., then 10x HS purif in (2′ @50° C.- 50 ul 2′ @42° C.) 197 1 1515 1:5 in house prot, 1836 1665 rGrG + G 1 SSRTII 12 90′ @42° 1 —Phusion 50 — — PCR w/o C., then 10x HS purif in (2′ @50° C.- 50 ul 2′@42° C.) 198 1 15 15 1:5 in house prot, 2542 1771 rGrG + G 1 SSRTII 1290′ @42° 1 — KAPA 50 — — PCR w/o C., then 10x HiFi HS purif in (2′ @50°C.- 50 ul 2′ @42° C.) 199 1 15 15 1:5 in house prot, 2393 1808 rGrG + G1 SSRTII 12 90′ @42° 1 — KAPA 50 — — PCR w/o C., then 10x HiFi HS purifin (2′ @50° C.- 50 ul 2′ @42° C.) 200 1 15 15 1:5 in house prot, 22001933 rGrG + G 1 SSRTII 12 90′ @42° 1 — Advantage 50 — — PCR w/o C., then10x purif in (2′ @50° C.- 50 ul 2′ @42° C.) 201 1 15 15 1:5 in houseprot, 2371 1920 rGrG + G 1 SSRTII 12 90′ @42° 1 — Advantage 50 — — PCRw/o C., then 10x purif in (2′ @50° C.- 50 ul 2′ @42° C.) 202 1 15 15 1:5in house prot, 1717 1780 rGrG + G 1 SSRTII 12 90′ @42° 1 — Q5 NEB 50 — —PCR w/o C., then 10x purif in (2′ @50° C.- 50 ul 2′ @42° C.) 203 1 15 151:5 in house prot, 1868 1759 rGrG + G 1 SSRTII 12 90′ @42° 1 — Q5 NEB 50— — PCR w/o C., then 10x purif in (2′ @50° C.- 50 ul 2′ @42° C.) 204 115 15 1:5 in house prot 40 1772 rGrG + G 1 SSRTIII 12 90′ @42° 1 yesAdvantage 50 — — C., then 10x (2′ @50° C.- 2′ @42° C.) 205 1 15 15 1:5in house prot 16 1017 rGrG + G 1 SSRTIII 12 90′ @42° 1 yes Advantage 50— — C., then 10x (2′ @50° C.- 2′ @42° C.) 206 1 15 15 1:5 in house prot88 1753 rGrG + G 1 SSRTIII 12 90′ @42° 1 yes Advantage 50 — — C., then10x (2′ @50° C.- 2′ @42° C.) 207 1 15 15 1:5 in house prot 61 1557rGrG + G 1 SSRTIII 12 90′ @42° 1 yes Advantage 50 — — C., then 10x (2′@50° C.- 2′ @42° C.) 208 1 15 15 1:5 in house prot 182 1766 rGrG + G 1SSRTIII 12 90′ @42° 1 yes Advantage 50 — — C., then 10x (2′ @50° C.- 2′@42° C.) 209 1 15 15 1:5 in house prot 136 1742 rGrG + G 1 SSRTIII 1290′ @42° 1 yes Advantage 50 — — C., then 10x (2′ @50° C.- 2′ @42° C.)210 1 15 15 1:5 in house prot 279 1701 rGrG + G 1 SSRTIII 12 90′ @42° 1yes Advantage 50 — — C., then 10x (2′ @50° C.- 2′ @42° C.) 211 1 15 151:5 in house prot 233 1643 rGrG + G 1 SSRTIII 12 90′ @42° 1 yesAdvantage 50 — — C., then 10x (2′ @50° C.- 2′ @42° C.) 212 1 15 15 1:5in house prot 447 1602 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50— — C., then 10x (2′ @50° C.- 2′ @42° C.) 213 1 15 15 1:5 in house prot363 1541 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then10x (2′ @50° C.- 2′ @42° C.) 214 1 15 15 1:5 in house prot 1279 1919rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then 10x (2′@50° C.- 2′ @42° C.) 215 1 15 15 1:5 in house prot 1739 1959 rGrG + G 1SSRTII 12 90′ @42° 1 yes Advantage 50 — — C., then 10x (2′ @50° C.- 2′@42° C.) 216 1 15 15 1:5 in house prot 1861 2063 rGrG + G 1 SSRTII 1290′ @42° 1 yes Advantage 50 — — C., then 10x (2′ @50° C.- 2′ @42° C.)217 1 15 15 1:5 in house prot 2329 2260 rGrG + G 1 SSRTII 12 90′ @42° 1yes Advantage 50 yes — (40 u C., then 10x SSRTII) (2′ @50° C.- 2′ @42°C.) 218 1 15 15 1:5 in house prot 2273 2218 rGrG + G 1 SSRTII 12 90′@42° 1 yes Advantage 50 yes — (40 u C., then 10x SSRTII) (2′ @50° C.- 2′@42° C.) 219 1 15 15 1:5 in house prot 2013 2268 rGrG + G 1 SSRTII 1290′ @42° 1 yes Advantage 50 yes — (40 u C., then 10x SSRTII, with (2′@50° C.- dNTPs + 2′ @42° C.) betaine added in the beginning) 220 1 15 151:5 in house prot 1932 2122 rGrG + G 1 SSRTII 12 90′ @42° 1 yesAdvantage 50 yes — (40 u C., then 10x SSRTII, with (2′ @50° C.- dNTPs +2′ @42° C.) betaine added in the beginning) 221 1 15 15 1:5 in houseprot 1877 2009 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes —(40 u C., then 10x SSRTII, with (2′ @50° C.- dNTPs + 2′ @42° C.)betaine + MgCl2 added in the beginning) 222 1 15 15 1:5 in house prot989 1963 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes — (40 uC., then 10x SSRTII, with (2′ @50° C.- dNTPs + 2′ @42° C.) betaine +MgCl2 added in the beginning) 223 1 15 15 1:5 in house prot 2269 1316rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes — (40 u C., then10x SSRTII, with (2′ @50° C.- all reagents 2′ @42° C.) except SSRTIIadded in the beginning) 224 1 15 15 1:5 in house prot 1926 1292 rGrG + G1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes — (40 u C., then 10x SSRTII,with (2′ @50° C.- all reagents 2′ @42° C.) except SSRTII added in thebeginning) 225 1 15 15 1:5 in house prot 1536 1297 rGrG + G 1 SSRTII 1290′ @42° 1 yes Advantage 50 yes — (40 u C., then 10x SSRTII, with (2′@50° C.- all reagents 2′ @42° C.) except SSRTII added in the beginning)226 1 15 15 1:5 in house prot 1604 1925 rGrG + G 1 SSRTII 12 90′ @42° 1yes Advantage 50 yes — C., then 10x (2′ @50° C.- 2′ @42° C.) 227 1 15 151:5 in house prot 1493 1910 rGrG + G 1 SSRTII 12 90′ @42° 1 yesAdvantage 50 yes — C., then 10x (2′ @50° C.- 2′ @42° C.) 228 1 15 15 1:5in house prot 1540 1924 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50yes — C., then 10x (2′ @50° C.- 2′ @42° C.) 229 1 15 15 1:5 in houseprot 1946 2143 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes —C., then 10x (2′ @50° C.- 2′ @42° C.) 230 1 15 15 1:5 in house prot 16202100 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes — C., then 10x(2′ @50° C.- 2′ @42° C.) 231 1 15 15 1:5 in house prot 1724 2059 rGrG +G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes — C., then 10x (2′ @50°C.- 2′ @42° C.) 232 1 15 15 1:5 in house prot 1355 1813 rGrG + G 1SSRTII 12 90′ @42° 1 yes Advantage 50 — — (no C., then 10x denaturation(2′ @50° C.- step @72° 2′ @42° C.) C. for oligo-dT (incubated 5′ @RT))233 1 15 15 1:5 in house prot 1330 1810 rGrG + G 1 SSRTII 12 90′ @42° 1yes Advantage 50 — — (no C., then 10x denaturation (2′ @50° C.- step@72° 2′ @42° C.) C. for oligo-dT (incubated 5′ @RT)) 234 1 15 15 1:5 inhouse prot 1319 1795 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 —— (no C., then 10x denaturation (2′ @50° C.- step @72° 2′ @42° C.) C.for oligo-dT (incubated 5′ @RT)) 235 1 15 15 1:5 in house prot 1493 1842rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes — (no C., then 10xdenaturation (2′ @50° C.- step @72° 2′ @42° C.) C. for oligo-dT(incubated 5′ @RT)) 236 1 15 15 1:5 in house prot 1332 1792 rGrG + G 1SSRTII 12 90′ @42° 1 yes Advantage 50 yes — (no C., then 10xdenaturation (2′ @50° C.- step @72° 2′ @42° C.) C. for oligo-dT(incubated 5′ @RT)) 237 1 15 15 1:5 in house prot 1728 2214 rGrG + G 1SSRTII 12 90′ @42° 1 yes Advantage 50 yes — C., then 10x (2′ @50° C.- 2′@42° C.) 238 1 15 15 1:5 in house prot 1620 2283 rGrG + G 1 SSRTII 1290′ @42° 1 yes Advantage 50 yes — C., then 10x (2′ @50° C.- 2′ @42° C.)239 1 15 15 1:5 in house prot 1563 2260 rGrG + G 1 SSRTII 12 90′ @42° 1yes Advantage 50 yes — C., then 10x (2′ @50° C.- 2′ @42° C.) 240 1 15 151:5 in house prot 1485 2171 rGrG + G 1 SSRTII 12 90′ @42° 1 yesAdvantage 50 yes — C., then 10x (2′ @55° C.- 2′ @42° C.) 241 1 15 15 1:5in house prot 1444 2281 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50yes — C., then 10x (2′ @55° C.- 2′ @42° C.) 242 1 15 15 1:5 in houseprot 1308 2231 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes —C., then 10x (2′ @55° C.- 2′ @42° C.) 243 1 15 15 1:5 in house prot 16182176 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes — C., then 10x(2′ @60° C.- 2′ @42° C.) 244 1 15 15 1:5 in house prot 1385 2168 rGrG +G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes — C., then 10x (2′ @60°C.- 2′ @42° C.) 245 1 15 15 1:5 in house prot 1588 2173 rGrG + G 1SSRTII 12 90′ @42° 1 yes Advantage 50 yes — C., then 10x (2′ @60° C.- 2′@42° C.) 246 1 15 15 1:5 in house prot 1332 2096 rGrG + G 1 SSRTII 1290′ @ 1 yes Advantage 50 yes — 42° C. 247 1 15 15 1:5 in house prot 13762054 rGrG + G 1 SSRTII 12 90′ @ 1 yes Advantage 50 yes — 42° C. 248 1 1515 1:5 in house prot 15 2251 rGrG + G 1 Maxima 4 90′ @ — yes Advantage50 yes — H− 55° C. 249 1 15 15 1:5 in house prot 22 1988 rGrG + G 1Maxima 4 90′ @ — yes Advantage 50 yes — H− 55° C. 250 1 15 15 1:5 inhouse prot 62 2084 rGrG + G 1 Maxima 4 90′ @ 1 yes Advantage 50 yes — H−55° C. 251 1 15 15 1:5 in house prot 84 1867 rGrG + G 1 Maxima 4 90′ @ 1yes Advantage 50 yes — H− 55° C. 252 1 15 15 1:5 in house prot 633 2065rGrG + G 1 Maxima 4 90′ @ — yes Advantage 50 yes — H− 55° C. 253 inhouse prot FAILED rGrG + G 1 Maxima 4 90′ @ — yes Advantage 50 yes —(+extra DTT) H− 60° C. 254 in house prot FAILED rGrG + G 1 Maxima 4 90′@ 1 yes Advantage 50 yes — (+extra DTT) H− 60° C. 255 in house protFAILED rGrG + G 1 Maxima 12 90′ @ 1 yes Advantage 50 yes — (+extra DTT)H− 60° C. 256 1 15 15 1:5 in house prot 80 1933 rGrG + G 1 Maxima 12 90′@ 1 yes Advantage 50 yes — (+extra DTT) H− 60° C. 257 1 15 15 1:5 inhouse prot 634 2177 rGrG + G 1 Maxima 4 90′ @ — yes Advantage 50 yes —(+extra DTT) H− 50° C. 258 1 15 15 1:5 in house prot 684 2195 rGrG + G 1Maxima 4 90′ @ — yes Advantage 50 yes — (+extra DTT) H− 50° C. 259 1 1515 1:5 in house prot 1013 2142 rGrG + G 1 Maxima 4 90′ @ 1 yes Advantage50 yes — (+extra DTT) H− 50° C. 260 1 15 15 1:5 in house prot 1195 2100rGrG + G 1 Maxima 4 90′ @ 1 yes Advantage 50 yes — (+extra DTT) H− 50°C. 261 1 15 15 1:5 in house prot 2192 1858 rGrG + G 1 Maxima 12 90′ @ 1yes Advantage 50 yes — (+extra DTT) H− 50° C. 262 1 15 15 1:5 in houseprot 2209 1837 rGrG + G 1 Maxima 12 90′ @ 1 yes Advantage 50 yes —(+extra DTT) H− 50° C. 263 1 15 15 1:5 in house prot 647 2117 rGrG + G 1Maxima 4 90′ @ — yes Advantage 50 yes — (NO H− 50° C. extra DTT) 264 115 15 1:5 in house prot 613 2147 rGrG + G 1 Maxima 4 90′ @ — yesAdvantage 50 yes — (NO H− 50° C. extra DTT) 265 1 15 15 1:5 in houseprot 1442 1989 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes —C., then 10x (2′ @50° C.- 2′ @42° C.) 266 1 15 15 1:5 in house prot 14962105 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes — C., then 10x(2′ @50° C.- 2′ @42° C.) 267 1 15 15 1:5 in house prot 1228 2677 rGrG +G 1 Maxima 4 90′ @ — — KAPA 50 yes — H− 50° C. HiFi HS 268 1 15 15 1:5in house prot 882 2283 rGrG + G 1 Maxima 4 90′ @ — — Advantage 50 yes —H− 50° C. 269 1 15 15 1:5 in house prot 2475 2558 rGrG + G 1 Maxima 490′ @ 1 — KAPA 50 yes — H− 50° C. HiFi HS 270 1 15 15 1:5 in house prot1557 2201 rGrG + G 1 Maxima 4 90′ @ 1 — Advantage 50 yes — H− 50° C. 2711 15 15 1:5 in house prot 4074 2185 rGrG + G 1 Maxima 12 90′ @ 1 — KAPA50 yes — H− 50° C. HiFi HS 272 1 15 15 1:5 in house prot 2927 2330rGrG + G 1 Maxima 12 90′ @ 1 — Advantage 50 yes — H− 50° C. 273 1 15 151:5 in house prot 3213 2524 rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA 50yes — C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 274 1 15 15 1:5 inhouse prot 3359 2662 rGrG + G 1 SSRTII 12 90′ @42° 1 — Advantage 50 yes— C., then 10x (2′ @50° C.- 2′ @42° C.) 275 1 15 15 1:5 in house prot819 2069 rGrG + G 1 Maxima 4 90′ @ — yes Advantage 50 yes — H− 50° C.276 1 15 15 1:5 in house prot 737 2202 rGrG + G 1 Maxima 4 90′ @ — yesAdvantage 50 yes — H− 50° C. 277 1 15 15 1:5 in house prot 1375 1937rGrG + G 1 Maxima 4 90′ @ 1 yes Advantage 50 yes — H− 50° C. 278 1 15 151:5 in house prot 1426 2008 rGrG + G 1 Maxima 4 90′ @ 1 yes Advantage 50yes — H− 50° C. 279 1 15 15 1:5 in house prot 2773 1848 rGrG + G 1Maxima 12 90′ @ 1 yes Advantage 50 yes — H− 50° C. 280 1 15 15 1:5 inhouse prot 2987 1916 rGrG + G 1 Maxima 12 90′ @ 1 yes Advantage 50 yes —H− 50° C. 281 1 15 15 1:5 in house prot 2155 2266 rGrG + G 1 SSRTII 1290′ @42° 1 yes Advantage 50 yes — C., then 10x (2′ @50° C.- 2′ @42° C.)282 1 15 15 1:5 in house prot 2240 2271 rGrG + G 1 SSRTII 12 90′ @42° 1yes Advantage 50 yes — C., then 10x (2′ @50° C.- 2′ @42° C.) 283 1 15 151:5 in house prot 694 2244 rGrG + G 1 SSRTII 6 90′ @42° — yes Advantage50 yes 1M C., then 10x proline (2′ @50° C.- 2′ @42° C.) 284 1 15 15 1:5in house prot 682 2361 rGrG + G 1 SSRTII 6 90′ @42° — yes Advantage 50yes 1M C., then 10x proline (2′ @50° C.- 2′ @42° C.) 285 1 15 15 1:5 inhouse prot 745 2264 rGrG + G 1 SSRTII 6 90′ @42° — yes Advantage 50 yes1M C., then 10x proline (2′ @50° C.- 2′ @42° C.) 286 1 15 15 1:5 inhouse prot 766 2311 rGrG + G 1 SSRTII 12 90′ @42° — yes Advantage 50 yes1M C., then 10x proline (2′ @50° C.- 2′ @42° C.) 287 1 15 15 1:5 inhouse prot 685 2304 rGrG + G 1 SSRTII 12 90′ @42° — yes Advantage 50 yes1M C., then 10x proline (2′ @50° C. 2′ @42° C.) 288 1 15 15 1:5 in houseprot 642 2226 rGrG + G 1 SSRTII 12 90′ @42° — yes Advantage 50 yes 1MC., then 10x proline (2′ @50° C.- 2′ @42° C.) 289 1 15 15 1:5 in houseprot 1133 1873 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes C.,then 10x (2′ @50° C.- 2′ @42° C.) 290 1 15 15 1:5 in house prot 14432148 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes C., then 10x(2′ @50° C.- 2′ @42° C.) 291 1 15 15 1:5 in house prot 1147 2066 rGrG +G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes C., then 10x (2′ @50° C.-2′ @42° C.) 292 1 15 15 1:5 in house prot 1322 2084 rGrG + G 1 SSRTII 1290′ @42° 1 yes Advantage 50 yes C., then 10x (2′ @50° C.- 2′ @42° C.)293 1 15 15 1:5 in house prot 1013 2095 rGrG + G 1 SSRTII 12 90′ @42° 1yes Advantage 50 yes C., then 10x (2′ @50° C.- 2′ @42° C.) 294 1 15 151:5 in house prot 960 2125 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage50 yes C., then 10x (2′ @50° C.- 2′ @42° C.) 295 1 15 15 1:5 in houseprot 1296 2129 rGrG + G 1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes withC., then 10x SMARTer (2′ @50° C.- dT30 2′ @42° C.) (unanchored oligo)296 1 15 15 1:5 in house prot 1390 2194 rGrG + G 1 SSRTII 12 90′ @42° 1yes Advantage 50 yes with C., then 10x SMARTer (2′ @50° C.- dT30 2′ @42°C.) (unanchored oligo) 297 1 15 15 1:5 in house prot 1419 2084 rGrG + G1 SSRTII 12 90′ @42° 1 yes Advantage 50 yes with C., then 10x SMARTer(2′ @50° C.- dT30 2′ @42° C.) (unanchored oligo) 298 1 18 15  1:20 inhouse prot 5456 2293 rGrG + G 2 SSRTII 12 90′ @42° 1 yes Advantage 50yes C., then 10x (2′ @50° C.- 2′ @42° C.) 299 1 18 15  1:20 in houseprot 5753 2334 rGrG + G 2 SSRTII 12 90′ @42° 1 yes Advantage 50 yes C.,then 10x (2′ @50° C.- 2′ @42° C.) 300 1 18 15  1:20 in house prot 53412365 rGrG + G 2 SSRTII 12 90′ @42° 1 yes Advantage 50 yes C., then 10x(2′ @50° C.- 2′ @42° C.) 301 1 18 15  1:20 in house prot 2266 1750rGrG + G 2 SSRTII 12 90′ @42° 1 yes Advantage 50 yes C., then 10x (2′@50° C.- 2′ @42° C.) 302 1 18 15  1:20 in house prot 1530 1850 rGrG + G2 SSRTII 12 90′ @42° 1 yes Advantage 50 yes C., then 10x (2′ @50° C.- 2′@42° C.) 303 1 18 15  1:20 in house prot 1883 1703 rGrG + G 2 SSRTII 1290′ @42° 1 yes Advantage 50 yes C., then 10x (2′ @50° C.- 2′ @42° C.)304 1 18 15  1:20 in house prot 2856 1731 rGrG + G 2 SSRTII 12 90′ @42°1 yes Advantage 50 yes C., then 10x (2′ @50° C.- 2′ @42° C.) 305 1 18 15 1:20 in house prot 3150 2371 rGrG + N 1 SSRTII 12 90′ @42° 1 yesAdvantage 50 yes C., then 10x (2′ @50° C.- 2′ @42° C.) 306 1 18 15  1:20in house prot 2889 2393 rGrG + N 1 SSRTII 12 90′ @42° 1 yes Advantage 50yes C., then 10x (2′ @50° C.- 2′ @42° C.) 307 1 18 15  1:20 in houseprot 3773 2302 rGrG + N 2 SSRTII 12 90′ @42° 1 yes Advantage 50 yes C.,then 10x (2′ @50° C.- 2′ @42° C.) 308 1 18 15  1:20 in house prot 37442318 rGrG + N 2 SSRTII 12 90′ @42° 1 yes Advantage 50 yes C., then 10x(2′ @50° C.- 2′ @42° C.) 309 1 18 15  1:20 in house prot 4113 2390rGrG + N 2 SSRTII 12 90′ @42° 1 yes Advantage 50 yes C., then 10x (2′@50° C.- 2′ @42° C.) 310 1 18 15  1:20 in house prot 3903 2309 rGrG + N2 SSRTII 12 90′ @42° 1 yes Advantage 50 yes C., then 10x (2′ @50° C.- 2′@42° C.) 311 1 18 15  1:20 in house prot 2649 2436 rGrG + N 2 SSRTII 1290′ @42° 1 yes Advantage 50 yes C., then 10x (2′ @50° C.- 2′ @42° C.)312 1 18 15  1:20 in house prot 2856 2422 rGrG + N 2 SSRTII 12 90′ @42°1 yes Advantage 50 yes C., then 10x (2′ @50° C.- 2′ @42° C.) 313 1 18 15 1:20 in house prot 2674 2412 rGrG + N 2 SSRTII 12 90′ @42° 1 yesAdvantage 50 yes C., then 10x (2′ @50° C.- 2′ @42° C.) 314 1 18 15  1:20in house prot 2641 2427 rGrG + N 2 SSRTII 12 90′ @42° 1 yes Advantage 50yes C., then 10x (2′ @50° C.- 2′ @42° C.) 315 1 18 15  1:20 in houseprot 5251 2272 rGrG + G 2 SSRTII 12 90′ @42° 1 yes Advantage 50 yes C.,then 10x (2′ @50° C.- 2′ @42° C.) 316 1 15 15 1:5 in house prot 12552057 rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA 50 yes C., then 10x HiFi HS(2′ @50° C.- 2′ @42° C.) 317 1 15 15 1:6 in house prot 1135 2123 rGrG +G 1 SSRTII 12 90′ @42° 1 — KAPA 50 yes C., then 10x HiFi HS (2′ @50° C.-2′ @42° C.) 318 1 15 15 1:7 in house prot 925 2224 rGrG + G 1 SSRTII 1290′ @42° 1 — KAPA 50 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.)319 1 15 15 1:8 in house prot 790 2326 rGrG + G 1 SSRTII 12 90′ @42° 1 —KAPA 50 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 320 1 15 151:9 in house prot 9 2866 rGrG + G 1 SSRTII 12 30′ @42° 1 — KAPA 50 yes1M C., HiFi HS sorbitol + 10′ @50° 0.3M C., trehalose 10′ @55° C. 321 115 15  1:10 in house prot 17 2648 rGrG + G 1 SSRTII 12 30′ @42° 1 — KAPA50 yes 1M C., HiFi HS sorbitol + 10′ @50° 0.3M C., trehalose 10′ @55° C.322 1 15 15  1:11 in house prot 18 2744 rGrG + G 1 SSRTII 12 30′ @42° 1— KAPA 50 yes 1M C., HiFi HS sorbitol + 10′ @50° 0.3M C., trehalose 10′@55° C. 323 1 15 15  1:12 in house prot 14 2844 rGrG + G 1 SSRTII 12 30′@42° 1 — KAPA 50 yes 1M C., HiFi HS sorbitol + 10′ @50° 0.3M C.,trehalose 10′ @55° C. 324 1 15 15  1:13 in house prot 24 2546 rGrG + G 1SSRTII 12 30′ @42° 1 — KAPA 50 yes 0.75M C., HiFi HS sorbitol + 10′ @50°0.15M C., trehalose 10′ @55° C. 325 1 15 15  1:14 in house prot 30 2478rGrG + G 1 SSRTII 12 30′ @42° 1 — KAPA 50 yes 0.75M C., HiFi HSsorbitol + 10′ @50° 0.15M C., trehalose 10′ @55° C. 326 1 15 15  1:15 inhouse prot 40 2609 rGrG + G 1 SSRTII 12 30′ @42° 1 — KAPA 50 yes 0.75MC., HiFi HS sorbitol + 10′ @50° 0.15M C., trehalose 10′ @55° C. 327 1 1815  1:10 in house prot 3197 2061 rGrG + G 1 SSRTII 15 90′ @42° 1 — KAPA50 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 328 1 18 15  1:10in house prot 3085 2065 rGrG + G 1 SSRTII 15 90′ @42° 1 — KAPA 50 yesC., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 329 1 18 15  1:10 in houseprot 2627 2134 rGrG + G 1 SSRTII 15 90′ @42° 1 — KAPA 50 yes C., then10x HiFi HS (2′ @50° C.- 2′ @42° C.) 330 1 18 15  1:10 in house prot2750 2035 rGrG + G 1 SSRTII 15 90′ @42° 1 — KAPA 50 yes C., then 10xHiFi HS (2′ @50° C.- 2′ @42° C.) 331 1 18 15  1:10 in house prot 9801920 rGrG + G 1 SSRTII 3 90′ @42° 1 — KAPA 50 yes C., then 10x HiFi HS(2′ @50° C.- 2′ @42° C.) 332 1 18 15  1:10 in house prot 997 1863 rGrG +G 1 SSRTII 3 90′ @42° 1 — KAPA 50 yes C., then 10x HiFi HS (2′ @50° C.-2′ @42° C.) 333 1 18 15  1:10 in house prot 1055 1925 rGrG + G 1 SSRTII3 90′ @42° 1 — KAPA 50 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.)334 1 18 15  1:10 in house prot 938 1931 rGrG + G 1 SSRTII 3 90′ @42° 1— KAPA 50 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 335 1 18 15 1:10 in house prot 2192 2119 rGrG + G 1 SSRTII 6 90′ @42° 1 — KAPA 50yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 336 1 18 15  1:10 inhouse prot 2122 2046 rGrG + G 1 SSRTII 6 90′ @42° 1 — KAPA 50 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 337 1 18 15  1:10 in houseprot 2408 2079 rGrG + G 1 SSRTII 6 90′ @42° 1 — KAPA 50 yes C., then 10xHiFi HS (2′ @50° C.- 2′ @42° C.) 338 1 18 15  1:10 in house prot 18362109 rGrG + G 1 SSRTII 6 90′ @42° 1 — KAPA 50 yes C., then 10x HiFi HS(2′ @50° C.- 2′ @42° C.) 339 1 18 15  1:10 in house prot 2861 2098rGrG + G 1 SSRTII 9 90′ @42° 1 — KAPA 50 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 340 1 18 15  1:10 in house prot 2518 2086 rGrG + G1 SSRTII 9 90′ @42° 1 — KAPA 50 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 341 1 18 15  1:10 in house prot 2289 2135 rGrG + G 1 SSRTII 990′ @42° 1 — KAPA 50 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.)342 1 18 15  1:10 in house prot 2553 2168 rGrG + G 1 SSRTII 9 90′ @42° 1— KAPA 50 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 343 1 18 15 1:10 in house prot 2571 2134 rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA 50yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 344 1 18 15  1:10 inhouse prot 2691 2115 rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA 50 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 345 1 18 15  1:10 in houseprot 2348 2121 rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA 50 yes C., then10x HiFi HS (2′ @50° C.- 2′ @42° C.) 346 1 18 15  1:10 in house prot2046 2162 rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA 50 yes C., then 10xHiFi HS (2′ @50° C.- 2′ @42° C.) 347 1 15 15 1:5 in house prot 37 1918rGrG + G 1 SSRTII 3 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 348 1 15 15 1:5 in house prot 65 1888 rGrG + G 1SSRTII 3 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 349 1 15 15 1:5 in house prot 46 1976 rGrG + G 1 SSRTII 3 90′@42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 350 115 15 1:5 in house prot 39 1966 rGrG + G 1 SSRTII 3 90′ @42° 1 — KAPA 25yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 351 1 15 15 1:5 inhouse prot 44 1784 rGrG + G 1 SSRTII 3 90′ @42° 1 — KAPA 25 yes C., then10x HiFi HS (2′ @50° C.- 2′ @42° C.) 352 1 15 15 1:5 in house prot 951791 rGrG + G 1 SSRTII 3 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS(2′ @50° C.- 2′ @42° C.) 353 1 15 15 1:5 in house prot 80 1897 rGrG + G1 SSRTII 3 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 354 1 15 15 1:5 in house prot 281 1685 rGrG + G 1 SSRTII 6 90′@42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 355 115 15 1:5 in house prot 348 1753 rGrG + G 1 SSRTII 6 90′ @42° 1 — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 356 1 15 15 1:5 inhouse prot 204 1734 rGrG + G 1 SSRTII 6 90′ @42° 1 — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 357 1 15 15 1:5 in house prot210 1840 rGrG + G 1 SSRTII 6 90′ @42° 1 — KAPA 25 yes C., then 10x HiFiHS (2′ @50° C.- 2′ @42° C.) 358 1 15 15 1:5 in house prot 207 1853rGrG + G 1 SSRTII 6 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 359 1 15 15 1:5 in house prot 285 1801 rGrG + G 1SSRTII 6 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 360 1 15 15 1:5 in house prot 120 1942 rGrG + G 1 SSRTII 6 90′@42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 361 115 15 1:5 in house prot 201 1732 rGrG + G 1 SSRTII 6 90′ @42° 1 — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 362 1 15 15 1:5 inhouse prot 395 1808 rGrG + G 1 SSRTII 9 90′ @42° 1 — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 363 1 15 15 1:5 in house prot558 1887 rGrG + G 1 SSRTII 9 90′ @42° 1 — KAPA 25 yes C., then 10x HiFiHS (2′ @50° C.- 2′ @42° C.) 364 1 15 15 1:5 in house prot 429 1792rGrG + G 1 SSRTII 9 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 365 1 15 15 1:5 in house prot 340 1779 rGrG + G 1SSRTII 9 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 366 1 15 15 1:5 in house prot 511 1797 rGrG + G 1 SSRTII 9 90′@42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 367 115 15 1:5 in house prot 353 1783 rGrG + G 1 SSRTII 9 90′ @42° 1 — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 368 1 15 15 1:5 inhouse prot 365 1785 rGrG + G 1 SSRTII 9 90′ @42° 1 — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 369 1 15 15 1:5 in house prot333 1840 rGrG + G 1 SSRTII 9 90′ @42° 1 — KAPA 25 yes C., then 10x HiFiHS (2′ @50° C.- 2′ @42° C.) 370 1 15 15 1:5 in house prot 477 1818rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 371 1 15 15 1:5 in house prot 517 1754 rGrG + G 1SSRTII 12 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 372 1 15 15 1:5 in house prot 265 1917 rGrG + G 1 SSRTII 12 90′@42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 373 115 15 1:5 in house prot 349 1815 rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 374 1 15 15 1:5 inhouse prot 255 1763 rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 375 1 15 15 1:5 in house prot388 1760 rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA 25 yes C., then 10x HiFiHS (2′ @50° C.- 2′ @42° C.) 376 1 15 15 1:5 in house prot 387 1816rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 377 1 15 15 1:5 in house prot 635 1928 rGrG + G 1SSRTII 15 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 378 1 15 15 1:5 in house prot 515 1873 rGrG + G 1 SSRTII 15 90′@42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 379 115 15 1:5 in house prot 658 1989 rGrG + G 1 SSRTII 15 90′ @42° 1 — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 380 1 15 15 1:5 inhouse prot 602 1905 rGrG + G 1 SSRTII 15 90′ @42° 1 — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 381 1 15 15 1:5 in house prot412 1896 rGrG + G 1 SSRTII 15 90′ @42° 1 — KAPA 25 yes C., then 10x HiFiHS (2′ @50° C.- 2′ @42° C.) 382 1 15 15 1:5 in house prot 476 1958rGrG + G 1 SSRTII 15 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 383 1 15 15 1:5 in house prot 422 1853 rGrG + G 1SSRTII 15 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 384 1 15 15 1:5 in house prot 551 1876 rGrG + G 1 SSRTII 15 90′@42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 385 115 15 1:5 in house prot 1736 1698 rGrG + G 1 SSRTII 20 90′ @42° 1 — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 386 1 15 15 1:5 inhouse prot 1294 1750 rGrG + G 1 SSRTII 20 90′ @42° 1 — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 387 1 15 15 1:5 in house prot1160 1736 rGrG + G 1 SSRTII 20 90′ @42° 1 — KAPA 25 yes C., then 10xHiFi HS (2′ @50° C.- 2′ @42° C.) 388 1 15 15 1:5 in house prot 1245 1786rGrG + G 1 SSRTII 20 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 389 1 15 15 1:5 in house prot 1234 1733 rGrG + G 1SSRTII 25 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 390 1 15 15 1:5 in house prot 1654 1684 rGrG + G 1 SSRTII 2590′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.)391 1 15 15 1:5 in house prot 1327 1696 rGrG + G 1 SSRTII 25 90′ @42° 1— KAPA 25 yes C., then 10x HiFi HS (2′ @50° C. 2′ @42° C.) 392 1 15 151:5 in house prot 1713 1596 rGrG + G 1 SSRTII 25 90′ @42° 1 — KAPA 25yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 393 1 15 15 1:5 inhouse prot 1428 1667 rGrG + G 1 SSRTII 30 90′ @42° 1 — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 394 1 15 15 1:5 in house prot1274 1711 rGrG + G 1 SSRTII 30 90′ @42° 1 — KAPA 25 yes C., then 10xHiFi HS (2′ @50° C.- 2′ @42° C.) 395 1 15 15 1:5 in house prot 1471 1651rGrG + G 1 SSRTII 30 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 396 1 15 15 1:5 in house prot 1362 1653 rGrG + G 1SSRTII 30 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 397 1 15 15 1:5 in house prot 86 1996 rGrG + G 1 SSRTII 6 90′@42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 398 115 15 1:5 in house prot 66 2002 rGrG + G 1 SSRTII 6 90′ @42° 1 — KAPA 25yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 399 1 15 15 1:5 inhouse prot 92 1941 rGrG + G 1 SSRTII 6 90′ @42° 1 — KAPA 25 yes C., then10x HiFi HS (2′ @50° C.- 2′ @42° C.) 400 1 15 15 1:5 in house prot 861898 rGrG + G 1 SSRTII 6 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS(2′ @50° C.- 2′ @42° C.) 401 1 15 15 1:5 in house prot 85 2071 rGrG + G1 SSRTII 9 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 402 1 15 15 1:5 in house prot 75 2078 rGrG + G 1 SSRTII 9 90′@42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 403 115 15 1:5 in house prot 120 2081 rGrG + G 1 SSRTII 9 90′ @42° 1 — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 404 1 15 15 1:5 inhouse prot 150 1984 rGrG + G 1 SSRTII 9 90′ @42° 1 — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 405 1 15 15 1:5 in house prot132 2088 rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA 25 yes C., then 10x HiFiHS (2′ @50° C.- 2′ @42° C.) 406 1 15 15 1:5 in house prot 101 2139rGrG + G 1 SSRTII 12 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 407 1 15 15 1:5 in house prot 131 2221 rGrG + G 1SSRTII 12 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 408 1 15 15 1:5 in house prot 149 2083 rGrG + G 1 SSRTII 12 90′@42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 409 115 15 1:5 in house prot 169 2105 rGrG + G 1 SSRTII 15 90′ @42° 1 — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 410 1 15 15 1:5 inhouse prot 195 2020 rGrG + G 1 SSRTII 15 90′ @42° 1 — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 411 1 15 15 1:5 in house prot141 2042 rGrG + G 1 SSRTII 15 90′ @42° 1 — KAPA 25 yes C., then 10x HiFiHS (2′ @50° C.- 2′ @42° C.) 412 1 15 15 1:5 in house prot 196 2077rGrG + G 1 SSRTII 15 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 413 1 15 15 1:5 in house prot 123 2133 rGrG + G 1SSRTII 20 90′ @42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 414 1 15 15 1:5 in house prot 136 2042 rGrG + G 1 SSRTII 20 90′@42° 1 — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 415 115 15 1:5 in house prot 153 2008 rGrG + G 1 SSRTII 20 90′ @42° 1 — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 416 1 15 15 1:5 inhouse prot 166 2111 rGrG + G 1 SSRTII 20 90′ @42° 1 — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 417 1 15 15 1:5 in house prot259 2484 rGrG + G 1 SSRTII 6 90′ @42° — — KAPA 25 yes C., then 10x HiFiHS (2′ @50° C.- 2′ @42° C.) 418 1 15 15 1:5 in house prot 255 2505rGrG + G 1 SSRTII 6 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 419 1 15 15 1:5 in house prot 231 2493 rGrG + G 1SSRTII 6 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 420 1 15 15 1:5 in house prot 258 2469 rGrG + G 1 SSRTII 6 90′@42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 421 115 15 1:5 in house prot 226 2529 rGrG + G 1 SSRTII 6 90′ @42° — — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 422 1 15 15 1:5 inhouse prot 280 2402 rGrG + G 1 SSRTII 6 90′ @42° — — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 423 1 15 15 1:5 in house prot270 2449 rGrG + G 1 SSRTII 6 90′ @42° — — KAPA 25 yes C., then 10x HiFiHS (2′ @50° C.- 2′ @42° C.) 424 1 15 15 1:5 in house prot 249 2557rGrG + G 1 SSRTII 6 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 425 1 15 15 1:5 in house prot 423 1680 rGrG + G 1SSRTII 9 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 426 1 15 15 1:5 in house prot 433 2537 rGrG + G 1 SSRTII 9 90′@42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 427 115 15 1:5 in house prot 504 2571 rGrG + G 1 SSRTII 9 90′ @42° — — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 428 1 15 15 1:5 inhouse prot 483 2002 rGrG + G 1 SSRTII 9 90′ @42° — — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 429 1 15 15 1:5 in house prot585 2409 rGrG + G 1 SSRTII 9 90′ @42° — — KAPA 25 yes C., then 10x HiFiHS (2′ @50° C.- 2′ @42° C.) 430 1 15 15 1:5 in house prot 502 2591rGrG + G 1 SSRTII 9 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 431 1 15 15 1:5 in house prot 541 2533 rGrG + G 1SSRTII 9 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 432 1 15 15 1:5 in house prot 554 2555 rGrG + G 1 SSRTII 9 90′@42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 433 115 15 1:5 in house prot 677 2368 rGrG + G 1 SSRTII 12 90′ @42° — — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 434 1 15 15 1:5 inhouse prot 792 2287 rGrG + G 1 SSRTII 12 90′ @42° — — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 435 1 15 15 1:5 in house prot842 2325 rGrG + G 1 SSRTII 12 90′ @42° — — KAPA 25 yes C., then 10x HiFiHS (2′ @50° C.- 2′ @42° C.) 436 1 15 15 1:5 in house prot 737 2312rGrG + G 1 SSRTII 12 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 437 1 15 15 1:5 in house prot 831 2233 rGrG + G 1SSRTII 12 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 438 1 15 15 1:5 in house prot 780 2468 rGrG + G 1 SSRTII 12 90′@42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 439 115 15 1:5 in house prot 41 2536 rGrG + G 1 SSRTII 6 90′ @42° — — KAPA 25yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 440 1 15 15 1:5 inhouse prot 28 2646 rGrG + G 1 SSRTII 6 90′ @42° — — KAPA 25 yes C., then10x HiFi HS (2′ @50° C.- 2′ @42° C.) 441 1 15 15 1:5 in house prot 442702 rGrG + G 1 SSRTII 6 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS(2′ @50° C.- 2′ @42° C.) 442 1 15 15 1:5 in house prot 30 2504 rGrG + G1 SSRTII 6 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 443 1 15 15 1:5 in house prot 47 2664 rGrG + G 1 SSRTII 9 90′@42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 444 115 15 1:5 in house prot 54 2652 rGrG + G 1 SSRTII 9 90′ @42° — — KAPA 25yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 445 1 15 15 1:5 inhouse prot 70 2502 rGrG + G 1 SSRTII 9 90′ @42° — — KAPA 25 yes C., then10x HiFi HS (2′ @50° C.- 2′ @42° C.) 446 1 15 15 1:5 in house prot 802555 rGrG + G 1 SSRTII 9 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS(2′ @50° C.- 2′ @42° C.) 447 1 15 15 1:5 in house prot 122 2374 rGrG + G1 SSRTII 12 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.-2′ @42° C.) 448 1 15 15 1:5 in house prot 138 2368 rGrG + G 1 SSRTII 1290′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.)449 1 15 15 1:5 in house prot 105 2441 rGrG + G 1 SSRTII 12 90′ @42° — —KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 450 1 15 151:5 in house prot 108 2377 rGrG + G 1 SSRTII 12 90′ @42° — — KAPA 25 yesC., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 451 1 15 15 1:5 in houseprot 128 2113 rGrG + G 1 SSRTII 15 90′ @42° — — KAPA 25 yes C., then 10xHiFi HS (2′ @50° C.- 2′ @42° C.) 452 1 15 15 1:5 in house prot 147 2049rGrG + G 1 SSRTII 15 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′@50° C.- 2′ @42° C.) 453 1 15 15 1:5 in house prot 101 2127 rGrG + G 1SSRTII 15 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′@42° C.) 454 1 15 15 1:5 in house prot 108 2251 rGrG + G 1 SSRTII 15 90′@42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 455 115 15 1:5 in house prot 128 2007 rGrG + G 1 SSRTII 20 90′ @42° — — KAPA25 yes C., then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 456 1 15 15 1:5 inhouse prot 86 2104 rGrG + G 1 SSRTII 20 90′ @42° — — KAPA 25 yes C.,then 10x HiFi HS (2′ @50° C.- 2′ @42° C.) 457 1 15 15 1:5 in house prot98 2147 rGrG + G 1 SSRTII 20 90′ @42° — — KAPA 25 yes C., then 10x HiFiHS (2′ @50° C.- 2′ @42° C.) 458 1 15 15 1:5 in house prot 94 1963 rGrG +G 1 SSRTII 20 90′ @42° — — KAPA 25 yes C., then 10x HiFi HS (2′ @50° C.-2′ @42° C.) B. Analyses of experimental variables effecting cDNA libraryyield and length To evaluate the effect on cDNA yield and length of eachcomponent of the protocol, we compiled groups of replicates fromdifferent experiments that only differed in the experimental variableevaluated (column A). These experiments were used to compute Studentt-test p-values and Wilcoxon rank sum test p-values for both cDNA yieldand cDNA average length. Experi- cDNA Yield cDNA length mental Wilcoxon-mean mean t-test wilcoxon- mean mean Variable t-test test yield yieldcDNA test cDNA length length Number of Tested Variant 1 Variant 2 YieldYield (Var1) (Var2) length length (Var1) (Var2) replicates elution 20 302.69E−02 3.33E−01 166.79 56.535 2.43E−01 3.33E−01 2230.5 1930 2 volume(ul) TSO 2rGrG + G 3rGrG + G 3.01E−02 3.33E−01 83.025 138.225 1.50E−013.33E−01 1887 1722.5 2 TSO 2rGrG + G C6 amino 4.43E−02 3.33E−01 83.02536.1875 3.11E−01 3.33E−01 1887 1747 2 TSO 2rGrG + G rGrG + G 2.56E−043.33E−01 83.025 299.025 1.71E−01 3.33E−01 1887 1707 2 TSO 2rGrG + Gphosphate 1.12E−02 3.33E−01 83.025 44.0625 7.13E−01 1.00E+00 1887 1907 2TSO 2OMe ddC 2.00E−01 9.52E−02 117.39 62.7 8.95E−01 8.41E−01 1779.21795.8 5 TSO 2OMe rGrG + G 2.59E−02 7.94E−03 117.39 369.615 7.72E−021.51E−01 1779.2 1597 5 TSO 2OMe rGrGrG 7.68E−01 8.86E−01 123.675109.9313 8.19E−03 2.86E−02 1797.75 1416.5 4 TSO 2OMe SMARTer — — 69.22584.225 — — 1734 1652 1 Oligo IIA TSO 3rGrG + G C6 amino 4.11E−033.33E−01 138.225 36.1875 8.17E−01 1.00E+00 1722.5 1747 2 TSO 3rGrG + GrGrG + G 1.36E−03 1.00E−01 130.375 285.775 6.09E−01 1.00E+00 1691.33331646.3333 3 TSO 3rGrG + G phosphate 2.63E−02 3.33E−01 138.225 44.06253.09E−02 3.33E−01 1722.5 1907 2 TSO C6 amino rGrG + G 3.25E−03 3.33E−0136.1875 299.025 7.44E−01 6.67E−01 1747 1707 2 TSO C6 amino phosphate3.42E−01 3.33E−01 36.1875 44.0625 2.97E−01 3.33E−01 1747 1907 2 TSO ddCrGrG + G 1.36E−02 7.94E−03 62.7 369.615 1.53E−01 2.22E−01 1795.8 1597 5TSO ddC rGrGrG 2.16E−01 2.00E−01 66.4313 109.9313 6.64E−02 1.14E−011770.5 1416.5 4 TSO ddC SMARTer — — 30.075 84.225 — — 1463 1652 1 OligoIIA TSO dGCGGG dGCGGGp 2.63E−03 1.00E−01 47.3067 14.2533 9.78E−041.00E−01 2104.6667 2309.3333 3 TSO dGCGGG rGrGrG 2.21E−02 1.00E−0147.3067 150.6733 9.85E−01 7.00E−01 2104.6667 2107.3333 3 TSO dGCGGGrGrGrGp 1.32E−01 3.33E−01 48.33 35.64 2.38E−03 3.33E−01 2117.5 2588.5 2TSO dGCGGGp rGrGrG 1.37E−02 1.00E−01 14.253 150.6733 2.41E−01 1.00E−012309.3333 2107.3333 3 TSO dGCGGGp rGrGrGp 2.47E−03 3.33E−01 14.48 35.645.93E−02 3.33E−01 2317 2588.5 2 TSO rGrG + G phosphate 2.41E−03 3.33E−01299.025 44.0625 1.89E−01 3.33E−01 1707 1907 2 TSO rGrG + G rGrGrG6.55E−03 4.96E−04 235.7125 82.075 1.71E−01 2.14E−01 1588.8333 1713 12TSO rGrG + G SMARTer 2.74E−01 3.33E−01 197.6625 77.175 5.20E−02 3.33E−011444 1622.5 2 Oligo IIA TSO rGrGrG rGrGrGp 4.64E−04 2.86E−02 154.56532.765 2.56E−05 2.86E−02 2256.5 2583.5 4 TSO rGrGrG SMARTer 1.62E−013.33E−01 106.0125 77.175 4.67E−01 6.67E−01 1475.5 1622.5 2 Oligo IIAMgCl2 0 3 5.27E−01 4.00E−01 65.95 82.975 9.97E−01 1.00E+00 1588.66671589 3 concentra- tion (mM) MgCl2 0 12 6.31E−01 1.00E+00 75.3375107.2125 7.78E−01 1.00E+00 1511.5 1480 2 concentra- tion (mM) MgCl2 4 63.46E−01 6.67E−01 102.3 124.05 2.28E−01 3.33E−01 1517.5 1398 2concentra- tion (mM) MgCl2 4 9 2.52E−02 3.33E−01 102.3 211.875 5.08E−016.67E−01 1517.5 1563.5 2 concentra- tion (mM) MgCl2 4 12 9.44E−051.30E−04 91.395 198.795 3.07E−01 2.47E−01 1918.8 1753.9 10 concentra-tion (mM) MgCl2 4 15 3.32E−01 3.33E−01 102.3 130.875 3.81E−01 6.67E−011517.5 1360 2 concentra- tion (mM) MgCl2 6 9 5.94E−01 6.86E−01 154.5938178.1625 3.80E−03 2.86E−02 1390.75 1607 4 concentra- tion (mM) MgCl2 612 8.77E−01 7.30E−01 127.0167 132.9417 4.77E−01 2.58E−01 1709.77781845.8889 9 concentra- tion (mM) MgCl2 6 15 4.50E−01 8.41E−01 140.52113.79 7.04E−01 5.48E−01 1443 1477 5 concentra- tion (mM) MgCl2 9 128.71E−01 6.86E−01 178.1625 170.8125 8.05E−01 8.86E−01 1607 1582.25 4concentra- tion (mM) MgCl2 9 15 1.17E−01 2.00E−01 178.1625 124.70621.24E−01 2.00E−01 1607 1448 4 concentra- tion (mM) MgCl2 12 15 2.94E−013.43E−01 170.8125 124.7062 2.87E−01 3.43E−01 1582.25 1448 4 concentra-tion (mM) betaine 0 0.6 7.74E−01 6.86E−01 80.6063 73.725 3.70E−021.14E−01 1690.75 1540.5 4 concentra- tion (M) betaine 0 1 1.61E−011.65E−01 51.93 81.2025 3.55E−01 2.18E−01 2127.8 2006.9 10 concentra-tion (M) betaine 0.6 1 4.49E−01 1.00E+00 87.1 69.875 7.58E−01 1.00E+001618.3333 1596.6667 3 concentra- tion (M) betaine 1 1.5 7.73E−013.43E−01 101.215 86.115 8.82E−01 6.86E−01 2409.5 2430.5 4 concentra-tion (M) RT Maxima Revertaid 1.92E−02 3.33E−01 192 15.6375 1.66E−013.33E−01 1696 1603.5 2 enzyme H− Premium RT Revertaid SSRTII 2.47E−024.11E−02 148.2125 222.375 9.03E−02 1.32E−01 1423 1552.8333 6 enzyme H−RT SMARTscribe SSRTII — — 15.9 19.65 — — 1669 1683 1 enzyme RT SSRTIISSRTIII 1.21E−06 1.55E−04 252.3094 9.7031 8.59E−01 6.74E−01 1637.1251618.875 8 enzyme RT 60@42 C., 60@50 C., 1.47E−01 3.33E−01 73.95 43.055.70E−01 1.00E+00 1628 1577.5 2 protocol then then 90@60 C. 90@42 C. RT60@42 C., 90@42 C., 1.50E−01 3.33E−01 73.95 107.3625 3.70E−01 3.33E−011628 1719 2 protocol then 30@60 C., 90@60 C. then 30@42 C. RT 60@50 C.,90@42 C., 5.90E−02 2.00E−01 55.7437 110.7375 1.58E−01 1.46E−01 1515 16294 protocol then 30@60 C., 90@42 C. then 30@42 C. RT 90@42 C. 90@42 C.,3.09E−01 3.43E−01 173.1562 235.8 6.43E−01 4.86E−01 1877.5 1973 4protocol then 10x (2@50 C.- 2@42 C.) RT 90@42 C. 90@42 C., 6.72E−023.33E−01 101.55 109.8375 1.90E−01 3.33E−01 2075 2226 2 protocol then 10x(2@55 C.- 2@42 C.) RT 90@42 C. 90@42 C., 8.90E−01 4.00E−01 158.45170.475 6.57E−01 4.00E−01 1924.3333 2025.3333 3 protocol then 10x (2@60C.- 2@42 C.) RT 90@42 C. 90@42 C., 4.30E−01 6.67E−01 244.7625 278.13756.57E−01 6.67E−01 1680 1644.5 2 protocol then 15x (2@50 C.- 2@42 C.) RT90@42 C. 90@42 C., 8.51E−01 6.67E−01 244.7625 237.225 1.20E−01 3.33E−011680 1467 2 protocol then 20x (2@50 C.- 2@42 C.) RT 90@42 C. 90@42 C.,2.86E−01 3.33E−01 244.7625 297.7125 4.87E−01 6.67E−01 1680 1738.5 2protocol then 5x (2@50 C.- 2@42 C.) RT 90@42 C., 90@42 C., 5.04E−031.00E−01 165.375 105.925 5.96E−01 1.00E+00 2248.6667 2227.6667 3protocol then 10x then 10x (2@50 C.- (2@55 C.- 2@42 C.) 2@42 C.) RT90@42 C., 90@42 C., 5.23E−01 4.21E−01 206.295 175.17 9.27E−01 5.48E−012031.2 2015.4 5 protocol then 10x then 10x (2@50 C.- (2@60 C.- 2@42 C.)2@42 C.) RT 90@42 C., 90@42 C., 7.32E−01 1.00E+00 291.125 300.3 1.71E−012.00E−01 1722.3333 1639.6667 3 protocol then 10x then 15x (2@50 C.-(2@50 C.- 2@42 C.) 2@42 C.) RT 90@42 C., 90@42 C., 4.97E−01 4.00E−01246.875 196.375 7.32E−02 1.00E−01 1714 1521 3 protocol then 10x then 20x(2@50 C.- (2@50 C.- 2@42 C.) 2@42 C.) RT 90@42 C., 90@42 C., 9.92E−011.00E+00 291.125 291.25 5.65E−01 7.00E−01 1722.3333 1752.6667 3 protocolthen 10x then 5x (2@50 C.- (2@50 C.- 2@42 C.) 2@42 C.) RT 90@42 C.,90@42 C., 2.69E−01 4.00E−01 105.925 114.775 2.23E−01 4.00E−01 2227.66672172.3333 3 protocol then 10x then 10x (2@55 C.- (2@60 C.- 2@42 C.) 2@42C.) RT 90@42 C., 90@42 C., — — 286.2 285.3 — — 1732 1610 1 protocol then10x then 15x (2@60 C.- (2@50 C.- 2@42 C.) 2@42 C.) RT 90@42 C., 90@42C., — — 286.2 259.275 — — 1732 1525 1 protocol then 10x then 20x (2@60C.- (2@50 C.- 2@42 C.) 2@42 C.) RT 90@42 C., 90@42 C., — — 286.2 306.075— — 1732 1706 1 protocol then 10x then 5x (2@60 C.- (2@50 C.- 2@42 C.)2@42 C.) RT 90@42 C., 90@42 C., 2.94E−01 3.33E−01 278.1375 237.2251.47E−01 3.33E−01 1644.5 1467 2 protocol then 15x then 20x (2@50 C.-(2@50 C.- 2@42 C.) 2@42 C.) RT 90@42 C., 90@42 C., 7.35E−01 1.00E+00300.3 291.25 2.30E−02 1.00E−01 1639.6667 1752.6667 3 protocol then 15xthen 5x (2@50 C.- (2@50 C.- 2@42 C.) 2@42 C.) RT 90@42 C., 90@42 C.,1.90E−01 3.33E−01 237.225 297.7125 8.13E−02 3.33E−01 1467 1738.5 2protocol then 20x then 5x (2@50 C.- (2@50 C.- 2@42 C.) 2@42 C.) RT 90@50C. 90@55 C. 3.50E−03 7.94E−03 62.595 12.24 2.09E−01 1.51E−01 2146.2 20515 protocol RT 90@50 C. 90@60 C. — — 164.4 6 — — 1858 1933 1 protocol PCRAdvantage 2 KAPA 6.44E−01 7.55E−01 186.6375 171.0125 5.18E−01 5.90E−011929.4167 2029.5 12 enzyme Pol. HiFi HS PCR Advantage 2 Phusion 4.33E−014.86E−01 212.3625 182.6813 8.11E−01 3.43E−01 1701 1722.75 4 enzyme Pol.HS PCR Advantage 2 Q5 NEB 5.85E−02 2.86E−02 212.3625 133.8562 6.29E−013.43E−01 1701 1743.75 4 enzyme Pol. PCR KAPA Phusion 2.60E−01 3.43E−01223.725 182.6813 7.49E−01 8.86E−01 1702 1722.75 4 enzyme HiFi HS HS PCRKAPA Q5 NEB 2.99E−02 2.86E−02 223.725 133.8562 4.93E−01 8.86E−01 17021743.75 4 enzyme HiFi HS PCR Phusion Q5 NEB 1.25E−01 1.14E−01 182.6813133.8562 5.99E−01 8.86E−01 1722.75 1743.75 4 enzyme HS purifica- 0 yes6.77E−01 6.05E−01 186.85 203.225 3.44E−01 3.87E−01 2022.2222 1875.4444 9tion. dNTPs 0 yes 4.55E−03 1.37E−02 193.6781 134.3812 3.44E−04 5.55E−051709.4167 2008.0833 24 added in the beginning other 0 0.816M 6.89E−041.00E−01 291.125 103.95 3.50E−03 1.00E−01 1722.3333 1093 3 additives 1,2propandiol other 0 1.075M 1.82E−01 3.33E−01 299.025 178.4625 9.93E−023.33E−01 1707 1421.5 2 additives ethylene glycol other 0 3 mM 1.10E−013.33E−01 56.535 28.095 1.13E−01 3.33E−01 1930 1479.5 2 additives MnCl2other 0 6 mM 1.05E−01 3.33E−01 56.535 16.95 1.03E−01 3.33E−01 1930 12132 additives MnCl2 other 0.3M 0.6M 8.18E−01 6.86E−01 77.0625 72.35634.45E−01 4.86E−01 1679 1634.25 4 additives trehalose trehalose other0.816M 1.075M 2.82E−01 3.33E−01 104.4375 178.4625 3.99E−02 3.33E−01 10951421.5 2 additives 1,2 ethylene propandiol glycol other 3 mM 6 mM1.45E−01 3.33E−01 28.095 16.95 1.46E−01 3.33E−01 1479.5 1213 2 additivesMnCl2 MnCl2

TABLE S2 List of all template switching oligonucleotides tested. 5′-end3′-end TSO Sequence blocking 5′-end 3′-end blocking name (5′ → 3′)groups modifications modifications groups 2OMe AAGCAGTGGTATCAACGCAGAGT —— 3 Riboguanosines + 2O′-Methyl ACrGrGrGmG 1 Guanosine C6AAGCAGTGGTATCAACGCAGAGT — — 3 Riboguanosines Aminolink Amino ACATrGrGrGC6 ddC AAGCAGTGGTATCAACGCAGAGT — — 4 Riboguanosines + 1 — ACrGrGrGrGddCDideoxycytosine dGCG AAGCAGTGGTATCAACGCAGAGT — — 1 Guanosine +1 Cytosine + — GG ACGCGGG 3 Guanosines dGCG AAGCAGTGGTATCAACGCAGAGT — —1 Guanosine + 1 Cytosine + Phosphate GGp ACGCGGG 3 Guanosines ISOiGiCiGAAGCAGTGGTATCAACGCA Methyl isoGuanosine- 1 Riboguanosine + 1Phosphate GAGTACrGrCrGrGrG C5 isoCytosine- Ribocytosine +3 Riboguanosines rGr AAGCAGTGGTATCAACGCAGAGT — — 2 Riboguanosines +1 LNA- — G + G ACrGrG + G modified Guanosine rGr AAGCAGTGGTATCAACGCAGAGT— — 2 Riboguanosines + 1 LNA- — G + N ACrGrG + Nmodified nucleotide (any) + G + AAGCAGTGGTATCAACGCAGAGT — —3 LNA-modified Guanosines — G + G AC + G + G + G rG +AAGCAGTGGTATCAACGCAGAGT — — 2 LNA-modified Guanosines — G + G ACrG + G +G rGrGr AAGCAGTGGTATCAACGCAGAGT — — 3 Riboguanosines — G ACATrGrGrG rG3pAAGCAGTGGTATCAACGCAGAGT — — 3 Riboguanosines Phosphate ACATrGrGrGp rG5AAGCAGTGGTATCAACGCAGAGT — — 5 Riboguanosines — ACrGrGrGrGrG

avg amount TSO PCR conc size (ul of 10 uM RT oligo MgCl2 betainepurification PCR rxn vol sample cell type species (ng/ul) (bp) TSOsolution in 10 ul RT rxn) enzyme dT (mM) (M) after RT? enzyme (ul) notesHEK_2 HEK293T H. sapiens 10.778 1099 rGrG + G 1 SSRTII SMARTer 13 1 yesAdvantage 50 Could be a cell aggregate dT30VN HEK_3 HEK293T H. sapiens9.596 1142 rGrG + G 1 SSRTII SMARTer 14 1 yes Advantage 50 Could be acell aggregate dT30VN HEK_4 HEK293T H. sapiens 1.160 1098 rGrG + G 1SSRTII SMARTer 15 1 yes Advantage 50 dT30VN HEK_5 HEK293T H. sapiens0.590 1203 rGrG + G 1 SSRTII SMARTer 16 1 yes Advantage 50 dT30VN HEK_6HEK293T H. sapiens 2.210 1175 rGrG + G 1 SSRTII SMARTer 17 1 yesAdvantage 50 dT30VN HEK_7 HEK293T H. sapiens 0.410 1136 rGrG + G 1SSRTII SMARTer 18 1 yes Advantage 50 dT30VN HEK_8 HEK293T H. sapiens4.184 1146 rGrG + G 1 SSRTII SMARTer 19 1 yes Advantage 50 Could be acell aggregate dT30VN HEK_9 HEK293T H. sapiens 0.840 1141 rGrG + G 1SSRTII SMARTer 20 1 yes Advantage 50 dT30VN HEK_10 HEK293T H. sapiens1.130 1088 rGrG + G 1 SSRTII SMARTer 21 1 yes Advantage 50 dT30VN HEK_12HEK293T H. sapiens 9.095 1106 rGrG + G 1 SSRTII SMARTer 23 1 yesAdvantage 50 Could be a cell aggregate dT30VN HEK_13 HEK293T H. sapiens0.490 1017 rGrG + G 1 SSRTII SMARTer 24 1 yes Advantage 50 dT30VN HEK_14HEK293T H. sapiens 3.640 1046 rGrG + G 1 SSRTII SMARTer 25 1 yesAdvantage 50 Could be a cell aggregate dT30VN HEK_16 HEK293T H. sapiens17.225 1072 rGrG + G 1 SSRTII SMARTer 27 1 yes Advantage 50 Could be acell aggregate dT30VN HEK_SMRT_1 HEK293T H. sapiens 0.760 1429 SMARTer 1SMARTscribe SMARTer 6 1 yes Advantage 50 Oligo IIA dT30VN HEK_SMRT_2HEK293T H. sapiens 2.304 1411 SMARTer 1 SMARTscribe SMARTer 6 1 yesAdvantage 50 Could be a cell aggregate Oligo IIA dT30VN HEK_SMRT_3HEK293T H. sapiens 1.544 1424 SMARTer 1 SMARTscribe SMARTer 6 1 yesAdvantage 50 Oligo IIA dT30VN HEK_SMRT_4 HEK293T H. sapiens 0.422 1457SMARTer 1 SMARTscribe SMARTer 6 1 yes Advantage 50 Oligo IIA dT30VNHEK_18 HEK293T H. sapiens 0.208 1384 rGrGrG 1 SSRTII SMARTer 12 1 yesAdvantage 50 40 u SSRT II dT30VN HEK_19 HEK293T H. sapiens 0.512 1541rGrGrG 1 SSRTII SMARTer 12 1 yes Advantage 50 40 u SSRT II dT30VN HEK_20HEK293T H. sapiens 0.550 1614 rGrGrG 1 SSRTII SMARTer 12 1 yes Advantage50 40 u SSRT II dT30VN HEK_21 HEK293T H. sapiens 0.240 1492 rGrGrG 1SSRTII SMARTer 12 1 yes Advantage 50 40 u SSRT II dT30VN HEK_22 HEK293TH. sapiens 0.957 1222 rGrGrG 1 SSRTII SMARTer 12 1 yes Advantage 50 40 uSSRT II dT30VN HEK_23 HEK293T H. sapiens 0.872 1392 rGrGrG 1 SSRTIISMARTer 12 1 yes Advantage 50 40 u SSRT II dT30VN HEK_24 HEK293T H.sapiens 0.315 1326 rGrGrG 1 SSRTII SMARTer 12 1 yes Advantage 50 40 uSSRT II dT30VN HEK_25 HEK293T H. sapiens 0.462 1474 rGrG + G 1 SSRTIISMARTer 12 1 yes Advantage 50 dT30VN HEK_26 HEK293T H. sapiens 0.2481441 rGrG + G 1 SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_27HEK293T H. sapiens 0.244 1487 rGrG + G 1 SSRTII SMARTer 12 1 yesAdvantage 50 dT30VN HEK_28 HEK293T H. sapiens 0.262 1496 rGrG + G 1SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_29 HEK293T H. sapiens0.761 1402 rGrG + G 1 SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_31HEK293T H. sapiens 0.594 1583 rGrG + G 1 SSRTII SMARTer 12 1 yesAdvantage 50 dT30VN HEK_32 HEK293T H. sapiens 0.457 1347 rGrG + G 1SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_33 HEK293T H. sapiens0.473 1507 rGrG + G 1 SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_34HEK293T H. sapiens 0.315 1501 rGrG + G 1 SSRTII SMARTer 12 1 yesAdvantage 50 dT30VN HEK_35 HEK293T H. sapiens 0.744 1917 rGrG + G 1SSRTII SMARTer 12 1 — Advantage 50 dT30VN HEK_37 HEK293T H. sapiens1.531 1626 rGrG + G 1 SSRTII SMARTer 12 1 — Advantage 50 dT30VN HEK_38HEK293T H. sapiens 0.344 1870 rGrG + G 1 SSRTII SMARTer 12 1 — Advantage50 dT30VN HEK_39 HEK293T H. sapiens 0.453 1968 rGrG + G 1 SSRTII SMARTer12 1 — Advantage 50 dT30VN HEK_40 HEK293T H. sapiens 0.580 1958 rGrG + G1 SSRTII SMARTer 12 1 — Advantage 50 dT30VN HEK_41 HEK293T H. sapiens0.382 1202 rGrG + G 1 SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_42HEK293T H. sapiens 0.392 1288 rGrG + G 1 SSRTII SMARTer 12 1 yesAdvantage 50 dT30VN HEK_43 HEK293T H. sapiens 0.844 1319 rGrG + G 1SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_44 HEK293T H. sapiens0.582 1452 rGrG + G 1 SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_45HEK293T H. sapiens 0.682 1312 rGrG + G 1 SSRTII SMARTer 12 1 yesAdvantage 50 dT30VN HEK_46 HEK293T H. sapiens 0.692 1411 rGrG + G 1SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_47 HEK293T H. sapiens0.716 1337 rGrGrG 1 SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_48HEK293T H. sapiens 0.578 1391 rGrGrG 1 SSRTII SMARTer 12 1 yes Advantage50 dT30VN HEK_49 HEK293T H. sapiens 2.681 1440 rGrGrG 1 SSRTII SMARTer12 1 yes Advantage 50 Could be a cell aggregate dT30VN HEK_50 HEK293T H.sapiens 1.158 1507 rGrGrG 1 SSRTII SMARTer 12 1 yes Advantage 50 dT30VNHEK_51 HEK293T H. sapiens 0.710 1389 rGrGrG 1 SSRTII SMARTer 12 1 yesAdvantage 50 dT30VN HEK_52 HEK293T H. sapiens 1.050 1463 rGrGrG 1 SSRTIISMARTer 12 1 yes Advantage 50 dT30VN HEK_53 HEK293T H. sapiens 0.7141384 rGrGrG 1 SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_54 HEK293TH. sapiens 0.637 1509 rGrGrG 1 SSRTII SMARTer 12 1 yes Advantage 50dT30VN HEK_55 HEK293T H. sapiens 1.338 1873 rGrGrG 1 SSRTII SMARTer 12 1— KAPA 50 dT30VN HiFi HS HEK_56 HEK293T H. sapiens 1.258 1846 rGrGrG 1SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_57 HEK293T H. sapiens3.012 1798 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HSHEK_58 HEK293T H. sapiens 1.763 1734 rGrG + G 1 SSRTII SMARTer 12 1 —KAPA 50 dT30VN HiFi HS HEK_59 HEK293T H. sapiens 2.837 1833 rGrGrG 1SSRTII SMARTer 12 1 — Advantage 50 dT30VN HEK_60 HEK293T H. sapiens1.889 1758 rGrGrG 1 SSRTII SMARTer 12 1 — Advantage 50 dT30VN HEK_61HEK293T H. sapiens 3.733 1841 rGrG + G 1 SSRTII SMARTer 12 1 — Advantage50 dT30VN HEK_62 HEK293T H. sapiens 2.430 1849 rGrG + G 1 SSRTII SMARTer12 1 — Advantage 50 dT30VN HEK_63 HEK293T H. sapiens 0.758 1845 rGrGrG 1SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_64 HEK293T H. sapiens0.948 1907 rGrGrG 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_65HEK293T H. sapiens 1.364 1837 rGrGrG 1 SSRTII SMARTer 12 1 — KAPA 50dT30VN HiFi HS HEK_66 HEK293T H. sapiens 1.897 1821 rGrGrG 1 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_67 HEK293T H. sapiens 2.7211746 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_68HEK293T H. sapiens 5.123 1699 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50Could be a cell aggregate dT30VN HiFi HS HEK_69 HEK293T H. sapiens 2.4991892 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_70HEK293T H. sapiens 1.632 1715 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50dT30VN HiFi HS HEK_71 HEK293T H. sapiens 2.614 1438 rGrG + G 1 SSRTIISMARTer 12 1 yes Advantage 50 dT30VN HEK_72 HEK293T H. sapiens 1.3431535 rGrG + G 1 SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_73HEK293T H. sapiens 2.618 1570 rGrG + G 1 SSRTII SMARTer 12 1 yesAdvantage 50 dT30VN HEK_74 HEK293T H. sapiens 2.037 1611 rGrG + G 1SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_75 HEK293T H. sapiens1.862 1494 rGrGrG 1 SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_76HEK293T H. sapiens 0.560 1567 rGrGrG 1 SSRTII SMARTer 12 1 yes Advantage50 dT30VN HEK_77 HEK293T H. sapiens 1.609 1604 rGrGrG 1 SSRTII SMARTer12 1 yes Advantage 50 dT30VN HEK_78 HEK293T H. sapiens 0.748 1392 rGrGrG1 SSRTII SMARTer 12 1 yes Advantage 50 dT30VN HEK_79 HEK293T H. sapiens2.876 2093 rGrG + G 1 SSRTII SMARTer 12 1 — Advantage 50 dT30VN HEK_80HEK293T H. sapiens 6.017 1975 rGrG + G 1 SSRTII SMARTer 12 1 — Advantage50 Could be a cell aggregate dT30VN HEK_81 HEK293T H. sapiens 5.110 1834rGrG + G 1 SSRTII SMARTer 12 1 — Advantage 50 Could be a cell aggregatedT30VN HEK_82 HEK293T H. sapiens 2.659 1912 rGrG + G 1 SSRTII SMARTer 121 — KAPA 50 dT30VN HiFi HS HEK_83 HEK293T H. sapiens 3.820 1848 rGrG + G1 SSRTII SMARTer 12 1 — KAPA 50 Could be a cell aggregate dT30VN HiFi HSHEK_84 HEK293T H. sapiens 1.875 1941 rGrG + G 1 SSRTII SMARTer 12 1 —KAPA 50 dT30VN HiFi HS HEK_85 HEK293T H. sapiens 4.477 2003 rGrG + G 1SSRTII SMARTer 12 1 — KAPA 50 Could be a cell aggregate dT30VN HiFi HSHEK_86 HEK293T H. sapiens 4.162 1784 rGrG + G 1 SSRTII SMARTer 12 1 —Advantage 50 Could be a cell aggregate dT30VN HEK_87 HEK293T H. sapiens0.539 1964 rGrG + G 1 Maxima SMARTer 12 1 — KAPA 50 H minus dT30VN HiFiHS HEK_88 HEK293T H. sapiens 0.526 1860 rGrG + G 1 Maxima SMARTer 12 1 —KAPA 50 H minus dT30VN HiFi HS HEK_89 HEK293T H. sapiens 0.288 1881rGrG + G 1 Maxima SMARTer 12 1 — KAPA 50 H minus dT30VN HiFi HS HEK_90HEK293T H. sapiens 0.650 1560 rGrG + G 1 Maxima SMARTer 12 1 — KAPA 50 Hminus dT30 HiFi HS HEK_91 HEK293T H. sapiens 0.341 1852 rGrG + G 1Maxima SMARTer 12 1 — KAPA 50 H minus dT30 HiFi HS HEK_92 HEK293T H.sapiens 0.354 1783 rGrG + G 1 Maxima SMARTer 12 1 — KAPA 50 H minus dT30HiFi HS HEK_93 HEK293T H. sapiens 1.520 1939 rGrG + N 1 SSRTII SMARTer12 1 — KAPA 50 dT30VN HiFi HS HEK_94 HEK293T H. sapiens 1.722 1740rGrG + N 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_95 HEK293TH. sapiens 0.485 1740 rGrG + N 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VNHiFi HS HEK_96 HEK293T H. sapiens 1.090 1833 rGrG + N 1 SSRTII SMARTer12 1 — KAPA 50 dT30VN HiFi HS HEK_97 HEK293T H. sapiens 2.856 1731rGrG + N 2 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_98 HEK293TH. sapiens 2.776 1881 rGrG + N 2 SSRTII SMARTer 12 1 — KAPA 50 dT30VNHiFi HS HEK_99 HEK293T H. sapiens 1.954 1730 rGrG + N 2 SSRTII SMARTer12 1 — KAPA 50 dT30VN HiFi HS HEK_100 HEK293T H. sapiens 2.836 1909rGrG + N 2 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_101 HEK293TH. sapiens 2.266 1750 rGrG + G 2 SSRTII SMARTer 12 1 — KAPA 50 dT30VNHiFi HS HEK_102 HEK293T H. sapiens 1.530 1850 rGrG + G 2 SSRTII SMARTer12 1 — KAPA 50 dT30VN HiFi HS HEK_103 HEK293T H. sapiens 1.883 1703rGrG + G 2 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_105 HEK293TH. sapiens 4.105 1929 rGrG + G 2 SSRTII SMARTer 12 1 — KAPA 50 Could bea cell aggregate dT30VN HiFi HS HEK_106 HEK293T H. sapiens 2.580 2009rGrG + G 2 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_107 HEK293TH. sapiens 2.572 1910 rGrG + G 2 SSRTII SMARTer 12 1 — KAPA 50 dT30VNHiFi HS HEK_108 HEK293T H. sapiens 1.496 1973 rGrG + G 2 SSRTII SMARTer12 1 — KAPA 50 dT30VN HiFi HS HEK_109 HEK293T H. sapiens 3.459 1908rGrG + G 2 SSRTII SMARTer 12 1 — KAPA 50 Could be a cell aggregatedT30VN HiFi HS HEK_110 HEK293T H. sapiens 4.591 1921 rGrG + G 2 SSRTIISMARTer 12 1 — Advantage 50 Could be a cell aggregate dT30VN HEK_111HEK293T H. sapiens 3.841 1972 rGrG + G 2 SSRTII SMARTer 12 1 — Advantage50 Could be a cell aggregate dT30VN HEK_112 HEK293T H. sapiens 4.2791916 rGrG + G 2 SSRTII SMARTer 12 1 — Advantage 50 Could be a cellaggregate dT30VN HEK_113 HEK293T H. sapiens 2.123 1648 rGrG + G 2 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_114 HEK293T H. sapiens 2.5261845 rGrG + G 2 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_115HEK293T H. sapiens 2.084 1569 rGrG + N 2 SSRTII SMARTer 12 1 — KAPA 50dT30VN HiFi HS HEK_116 HEK293T H. sapiens 0.511 1628 rGrG + N 2 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_117 HEK293T H. sapiens 2.6901823 rGrG + N 2 SSRTII SMARTer 12 1 — Advantage 50 dT30VN HEK_118HEK293T H. sapiens 1.454 1774 rGrG + N 2 SSRTII SMARTer 12 1 — Advantage50 dT30VN HEK_119 HEK293T H. sapiens 0.361 1598 rGrG + N 2 SSRTIISMARTer 12 1 — Advantage 50 dT30VN HEK_120 HEK293T H. sapiens 1.215 1935rGrG + N 2 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_121 HEK293TH. sapiens 0.434 1682 rGrG + N 2 SSRTII SMARTer 12 1 — KAPA 50 dT30VNHiFi HS HEK_122 HEK293T H. sapiens 0.739 1553 rGrG + N 2 SSRTII SMARTer12 1 — KAPA 50 dT30VN HiFi HS HEK_123 HEK293T H. sapiens 1.599 1688rGrG + N 2 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_124 HEK293TH. sapiens 2.301 1617 rGrG + N 2 SSRTII SMARTer 12 1 — KAPA 50 dT30VNHiFi HS HEK_135 HEK293T H. sapiens 0.784 1810 rGrG + G 1 SSRTII SMARTer12 1 — KAPA 50 2x dCTP dT30VN HiFi HS HEK_136 HEK293T H. sapiens 1.3111990 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 2x dCTP dT30VN HiFi HSHEK_137 HEK293T H. sapiens 0.895 1829 rGrG + G 1 SSRTII SMARTer 12 1 —KAPA 50 2x dCTP dT30VN HiFi HS HEK_138 HEK293T H. sapiens 1.318 1948rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 2x dCTP dT30VN HiFi HS HEK_139HEK293T H. sapiens 1.853 1704 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 502x dCTP dT30VN HiFi HS HEK_140 HEK293T H. sapiens 4.009 1833 rGrG + G 1SSRTII SMARTer 12 1 — KAPA 50 2x dCTP (NOT SINGLE CELL) dT30VN HiFi HSHEK_141 HEK293T H. sapiens 5.230 1820 rGrG + G 1 SSRTII SMARTer 12 1 —KAPA 50 Could be a cell aggregate dT30VN HiFi HS HEK_144 HEK293T H.sapiens 4.803 1766 rGrG + G 2 SSRTII SMARTer 12 1 — Advantage 50 Couldbe a cell aggregate dT30VN HEK_145 HEK293T H. sapiens 2.166 1619 rGrG +G 2 SSRTII SMARTer 12 1 — Advantage 50 dT30VN HEK_146 HEK293T H. sapiens2.283 1552 rGrG + G 2 SSRTII SMARTer 12 1 — Advantage 50 dT30VN HEK_147HEK293T H. sapiens 2.099 1316 rGrG + G 2 SSRTII SMARTer 12 1 — Advantage50 dT30VN HEK_148 HEK293T H. sapiens 2.191 1339 rGrG + G 2 SSRTIISMARTer 12 1 — Advantage 50 HEDGEHOG PATTERN dT30VN HEK_149 HEK293T H.sapiens 1.560 1426 rGrG + G 2 SSRTII SMARTer 12 1 — Advantage 50 dT30VNHEK_150 HEK293T H. sapiens 2.830 1508 rGrG + G 2 SSRTII SMARTer 12 1 —Advantage 50 dT30VN HEK_151 HEK293T H. sapiens 2.220 1220 rGrG + G 2SSRTII SMARTer 12 1 — Advantage 50 dT30 HEK_152 HEK293T H. sapiens 2.6541534 rGrG + G 2 SSRTII SMARTer 12 1 — Advantage 50 HEDGEHOG PATTERN dT30HEK_153 HEK293T H. sapiens 2.039 1655 rGrG + G 2 SSRTII SMARTer 12 1 —Advantage 50 dT30 HEK_154 HEK293T H. sapiens 3.001 1837 rGrG + G 2SSRTII SMARTer 12 1 — Advantage 50 dT30 HEK_155 HEK293T H. sapiens 1.3781846 rGrG + G 2 SSRTII SMARTer 12 1 — Advantage 50 dT30 HEK_156 HEK293TH. sapiens 1.065 1853 rGrG + G 2 SSRTII SMARTer 12 1 — Advantage 50 dT30HEK_157 HEK293T H. sapiens 1.621 1772 rGrG + G 2 SSRTII SMARTer 12 1 —Advantage 50 dT30 HEK_158 HEK293T H. sapiens 2.418 1706 rGrG + G 2SSRTII SMARTer 12 1 — Advantage 50 dT30 HEK_SMRT_5 HEK293T H. sapiens0.128 1495 SMARTer 1 SMARTscribe SMARTer 6 1 yes Advantage 50 Oligo IIAdT30VN HEK_SMRT_6 HEK293T H. sapiens 0.042 1469 SMARTer 1 SMARTscribeSMARTer 6 1 yes Advantage 50 Oligo IIA dT30VN HEK_SMRT_7 HEK293T H.sapiens 0.207 1359 SMARTer 1 SMARTscribe SMARTer 6 1 yes Advantage 50Oligo IIA dT30VN HEK_SMRT_8 HEK293T H. sapiens 0.246 1340 SMARTer 1SMARTscribe SMARTer 6 1 yes Advantage 50 Oligo IIA dT30VN HEK_SMRT_9HEK293T H. sapiens 0.152 1455 SMARTer 1 SMARTscribe SMARTer 6 1 yesAdvantage 50 Oligo IIA dT30VN HEK_SMRT_10 HEK293T H. sapiens 0.256 1228SMARTer 1 SMARTscribe SMARTer 6 1 yes Advantage 50 Oligo IIA dT30VNHEK_SMRT_11 HEK293T H. sapiens 0.072 1238 SMARTer 1 SMARTscribe SMARTer6 1 yes Advantage 50 Oligo IIA dT30VN HEK_SMRT_12 HEK293T H. sapiens0.256 1292 SMARTer 1 SMARTscribe SMARTer 6 1 yes Advantage 50 Oligo IIAdT30VN C_1 C2C12 M. musculus 3.003 1696 rGrG + G 1 SSRTII SMARTer 12 1 —KAPA 25 Could be a cell aggregate dT30VN HiFi HS C_2 C2C12 M. musculus1.110 1772 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25 dT30VN HiFi HS C_3C2C12 M. musculus 1.157 1660 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25dT30VN HiFi HS C_4 C2C12 M. musculus 1.965 1674 rGrG + G 1 SSRTIISMARTer 12 1 — KAPA 25 dT30VN HiFi HS C_5 C2C12 M. musculus 2.884 1708rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25 Could be a cell aggregatedT30VN HiFi HS C_6 C2C12 M. musculus 2.325 1646 rGrG + G 1 SSRTIISMARTer 12 1 — KAPA 25 dT30VN HiFi HS C_7 C2C12 M. musculus 1.941 1577rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25 dT30VN HiFi HS C_8 C2C12 M.musculus 2.248 1666 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25 dT30VN HiFiHS C_9 C2C12 M. musculus 1.379 1614 rGrG + G 1 SSRTII SMARTer 12 1 —KAPA 50 dT30VN HiFi HS C_10 C2C12 M. musculus 1.832 1809 rGrG + G 1SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS C_11 C2C12 M. musculus3.644 1735 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 Could be a cellaggregate dT30VN HiFi HS C_12 C2C12 M. musculus 1.245 1735 rGrG + G 1SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS C_13 C2C12 M. musculus1.913 1896 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS C_14C2C12 M. musculus 1.703 1836 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50dT30VN HiFi HS C_15 C2C12 M. musculus 1.588 1937 rGrG + G 1 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS C_16 C2C12 M. musculus 1.730 1937rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS MEF_1 MEF M.musculus 0.999 1397 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25 dT30VN HiFiHS MEF_2 MEF M. musculus 2.862 1710 rGrG + G 1 SSRTII SMARTer 12 1 —KAPA 25 dT30VN HiFi HS MEF_3 MEF M. musculus 1.571 1577 rGrG + G 1SSRTII SMARTer 12 1 — KAPA 25 dT30VN HiFi HS MEF_4 MEF M. musculus 1.1201489 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25 dT30VN HiFi HS MEF_5 MEFM. musculus 2.121 1672 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25 dT30VNHiFi HS MEF_6 MEF M. musculus 5.025 1610 rGrG + G 1 SSRTII SMARTer 12 1— KAPA 25 Could be a cell aggregate dT30VN HiFi HS MEF_7 MEF M. musculus5.874 1065 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25 Could be a cellaggregate dT30VN HiFi HS MEF_8 MEF M. musculus 1.272 ? rGrG + G 1 SSRTIISMARTer 12 1 — KAPA 25 dT30VN HiFi HS MEF_9 MEF M. musculus 1.650 ?rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25 dT30VN HiFi HS MEF_10 MEF M.musculus 2.071 ? rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25 dT30VN HiFi HSMEF_11 MEF M. musculus 0.755 ? rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25dT30VN HiFi HS MEF_12 MEF M. musculus 7.528 ? rGrG + G 1 SSRTII SMARTer12 1 — KAPA 25 Could be a cell aggregate dT30VN HiFi HS MEF_13 MEF M.musculus 2.271 ? rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25 dT30VN HiFi HSMEF_14 MEF M. musculus 2.327 ? rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25dT30VN HiFi HS MEF_15 MEF M. musculus 3.149 ? rGrG + G 1 SSRTII SMARTer12 1 — KAPA 25 Could be a cell aggregate dT30VN HiFi HS MEF_16 MEF M.musculus 2.045 ? rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 25 dT30VN HiFi HSMEF_17 MEF M. musculus 0.497 1803 rGrGrG 1 SSRTII SMARTer 12 1 — KAPA 50dT30VN HiFi HS MEF_18 MEF M. musculus 0.427 1879 rGrGrG 1 SSRTII SMARTer12 1 — KAPA 50 dT30VN HiFi HS MEF_19 MEF M. musculus 0.406 1873 rGrGrG 1SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS MEF_20 MEF M. musculus0.680 1984 rGrGrG 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS MEF_21MEF M. musculus 0.429 1738 rGrGrG 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VNHiFi HS MEF_22 MEF M. musculus 0.634 2089 rGrGrG 1 SSRTII SMARTer 12 1 —KAPA 50 dT30VN HiFi HS MEF_23 MEF M. musculus 0.722 2019 rGrGrG 1 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS MEF_24 MEF M. musculus 0.397 1881rGrGrG 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS MEF_25 MEF M.musculus 0.981 2007 rGrG + G 1 SSRTII SMARTer 12 1 — Advantage 50 dT30VNMEF_26 MEF M. musculus 0.626 1827 rGrG + G 1 SSRTII SMARTer 12 1 —Advantage 50 dT30VN MEF_28 MEF M. musculus 0.656 1979 rGrG + G 1 SSRTIISMARTer 12 1 — Advantage 50 dT30VN MEF_29 MEF M. musculus 1.356 2143rGrG + G 1 SSRTII SMARTer 12 1 — Advantage 50 dT30VN MEF_30 MEF M.musculus 1.242 2137 rGrG + G 1 SSRTII SMARTer 12 1 — Advantage 50 dT30VNMEF_31 MEF M. musculus 2.240 2015 rGrG + G 1 SSRTII SMARTer 12 1 —Advantage 50 dT30VN MEF_32 MEF M. musculus 0.781 1817 rGrG + G 1 SSRTIISMARTer 12 1 — Advantage 50 dT30VN MEF_33 MEF M. musculus 0.603 1781rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS MEF_34 MEF M.musculus 5.014 1894 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 Could be acell aggregate dT30VN HiFi HS MEF_35 MEF M. musculus 1.651 2064 rGrG + G1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS MEF_36 MEF M. musculus1.017 1773 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HSMEF_37 MEF M. musculus 1.119 1783 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA50 dT30VN HiFi HS MEF_38 MEF M. musculus 0.681 1659 rGrG + G 1 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS MEF_39 MEF M. musculus 1.444 1469rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS MEF_40 MEF M.musculus 0.845 1401 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFiHS SMRT_1 C2C12 M. musculus 0.063 1170 SMARTer 1 SMARTscribe SMARTer 121 yes Advantage 50 Oligo IIA dT30VN SMRT_2 C2C12 M. musculus 0.165 1127SMARTer 1 SMARTscribe SMARTer 12 1 yes Advantage 50 Oligo IIA dT30VNSMRT_3 C2C12 M. musculus 0.130 1169 SMARTer 1 SMARTscribe SMARTer 12 1yes Advantage 50 Oligo IIA dT30VN SMRT_4 C2C12 M. musculus 0.091 1325SMARTer 1 SMARTscribe SMARTer 12 1 yes Advantage 50 Oligo IIA dT30VNSMRT_5 C2C12 M. musculus 0.036 1282 SMARTer 1 SMARTscribe SMARTer 12 1yes Advantage 50 Oligo IIA dT30VN SMRT_6 C2C12 M. musculus 0.135 1373SMARTer 1 SMARTscribe SMARTer 12 1 yes Advantage 50 Oligo IIA dT30VNSMRT_7 C2C12 M. musculus 0.161 1525 SMARTer 1 SMARTscribe SMARTer 12 1yes Advantage 50 Oligo IIA dT30VN SMRT_8 C2C12 M. musculus 0.178 1314SMARTer 1 SMARTscribe SMARTer 12 1 yes Advantage 50 Oligo IIA dT30VNSMRT_9 C2C12 M. musculus 0.036 1363 SMARTer 1 SMARTscribe SMARTer 12 1yes Advantage 50 Oligo IIA dT30VN SMRT_10 C2C12 M. musculus 0.129 1261SMARTer 1 SMARTscribe SMARTer 12 1 yes Advantage 50 Oligo IIA dT30VNSMRT_11 C2C12 M. musculus 0.080 1234 SMARTer 1 SMARTscribe SMARTer 12 1yes Advantage 50 Oligo IIA dT30VN SMRT_12 C2C12 M. musculus 0.111 1447SMARTer 1 SMARTscribe SMARTer 12 1 yes Advantage 50 Oligo IIA dT30VNSMRT_14 C2C12 M. musculus 0.081 1408 SMARTer 1 SMARTscribe SMARTer 12 1yes Advantage 50 Oligo IIA dT30VN SMRT_16 C2C12 M. musculus 0.074 1280SMARTer 1 SMARTscribe SMARTer 12 1 yes Advantage 50 Oligo IIA dT30VNBC_1 B-cells H. sapiens 0.826 1657 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA50 dT30VN HiFi HS BC_3 B-cells H. sapiens 1.091 1659 rGrG + G 1 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_4 B-cells H. sapiens 0.401 1649rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_5 B-cells H.sapiens 0.501 1547 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFiHS BC_6 B-cells H. sapiens 0.573 1534 rGrG + G 1 SSRTII SMARTer 12 1 —KAPA 50 dT30VN HiFi HS BC_7 B-cells H. sapiens 0.328 1343 rGrG + G 1SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_8 B-cells H. sapiens0.661 1607 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_9B-cells H. sapiens 0.500 1605 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50dT30VN HiFi HS BC_10 B-cells H. sapiens 0.840 1782 rGrG + G 1 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_11 B-cells H. sapiens 0.8291813 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_12B-cells H. sapiens 0.648 1642 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50dT30VN HiFi HS BC_13 B-cells H. sapiens 0.297 1815 rGrG + G 1 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_15 B-cells H. sapiens 0.9821825 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS HEK_159HEK293T H. sapiens 3.057 1464 rGrG + G 1 SSRTII SMARTer 15 1 — KAPA 25Could be a cell aggregate dT30VN HiFi HS HEK_160 HEK293T H. sapiens3.506 1799 rGrG + G 1 SSRTII SMARTer 15 1 — KAPA 25 Could be a cellaggregate dT30VN HiFi HS HEK_161 HEK293T H. sapiens 2.027 1611 rGrG + G1 SSRTII SMARTer 15 1 — KAPA 25 dT30VN HiFi HS HEK_162 HEK293T H.sapiens 2.513 1714 rGrG + G 1 SSRTII SMARTer 15 1 — KAPA 25 dT30VN HiFiHS HEK_163 HEK293T H. sapiens 0.558  909 rGrG + G 1 SSRTII SMARTer 20 1— KAPA 25 dT30VN HiFi HS HEK_164 HEK293T H. sapiens 1.490 1397 rGrG + G1 SSRTII SMARTer 20 1 — KAPA 25 dT30VN HiFi HS HEK_165 HEK293T H.sapiens 0.606 1334 rGrG + G 1 SSRTII SMARTer 20 1 — KAPA 25 dT30VN HiFiHS HEK_166 HEK293T H. sapiens 1.203 1515 rGrG + G 1 SSRTII SMARTer 20 1— KAPA 25 dT30VN HiFi HS BC_17 B-cells H. sapiens 0.393 1603 rGrG + G 1SSRTII SMARTer 12 — — KAPA 50 dT30VN HiFi HS BC_19 B-cells H. sapiens0.216 1730 rGrG + G 1 SSRTII SMARTer 12 — — KAPA 50 dT30VN HiFi HS BC_20B-cells H. sapiens 0.074 1131 rGrG + G 1 SSRTII SMARTer 12 — — KAPA 50dT30VN HiFi HS BC_21 B-cells H. sapiens 0.126 1445 rGrG + G 1 SSRTIISMARTer 12 — — KAPA 50 dT30VN HiFi HS BC_22 B-cells H. sapiens 0.3251702 rGrG + G 1 SSRTII SMARTer 12 — — KAPA 50 dT30VN HiFi HS BC_23B-cells H. sapiens 0.183 1756 rGrG + G 1 SSRTII SMARTer 12 — — KAPA 50dT30VN HiFi HS BC_24 B-cells H. sapiens 0.097 1338 rGrG + G 1 SSRTIISMARTer 12 — — KAPA 50 dT30VN HiFi HS BC_25 B-cells H. sapiens 0.8261657 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_26B-cells H. sapiens 1.091 1659 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50dT30VN HiFi HS BC_27 B-cells H. sapiens 0.401 1649 rGrG + G 1 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_28 B-cells H. sapiens 0.5011547 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_29B-cells H. sapiens 0.573 1534 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50dT30VN HiFi HS BC_30 B-cells H. sapiens 0.328 1343 rGrG + G 1 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_31 B-cells H. sapiens 0.6611607 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_32B-cells H. sapiens 0.500 1605 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50dT30VN HiFi HS BC_33 B-cells H. sapiens 0.840 1782 rGrG + G 1 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_34 B-cells H. sapiens 0.8291813 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_35B-cells H. sapiens 0.648 1642 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50dT30VN HiFi HS BC_36 B-cells H. sapiens 0.297 1815 rGrG + G 1 SSRTIISMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_37 B-cells H. sapiens 0.9821825 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50 dT30VN HiFi HS BC_38B-cells H. sapiens 0.853 1795 rGrG + G 1 SSRTII SMARTer 12 1 — KAPA 50dT30VN HiFi HS

TABLE S4 Detailing variants of the Smart-seq2 protocol Bead purificationProtocol amount TSO before pre- name TSO (ul) amplification? DNAPolymerase Smart- rGrG + G 1 ul (10 uM) no KAPA HiFi seq2 HotStartReadyMix Variant 1 rGrG + G 2 ul (10 uM) no KAPA HiFi HotStart ReadyMixVariant 2 rGrG + N 2 ul (10 uM) no KAPA HiFi HotStart ReadyMix Variant 3rGrG + G 1 ul (10 uM) no Advantage 2 Variant 4 rGrGrG 1 ul (10 uM) noKAPA HiFi HotStart ReadyMix SMARTer SMARTer 1 ul (12 uM) yes Advantage 2Oligo IIA

1-20. (canceled)
 21. A cDNA library produced by a method of comprisingthe steps of: annealing a cDNA synthesis primer to said RNA molecule andsynthesizing a first cDNA strand to form an RNA-cDNA intermediate; andconducting a reverse transcriptase reaction by contacting said RNA-cDNAintermediate with a template switching oligonucleotide (TSO), whereinthe TSO comprises a locked nucleic acid (LNA) at its 3′-end, underconditions suitable for extension of the first DNA strand that iscomplementary to the RNA molecule, rendering it additionallycomplementary to the TSO. 22-30. (canceled)
 31. The cDNA library ofclaim 21, wherein said reverse transcription reaction is conducted inthe presence of a methyl group donor and a metal salt.
 32. The cDNAlibrary of claim 31, wherein said methyl group donor is betaine.
 33. ThecDNA library of claim 31, wherein said metal salt is magnesium salt. 34.The cDNA library of claim 33, wherein said magnesium salt has aconcentration of at least 7 mM, at least 8 mM, or at least 9 mM.
 35. ThecDNA library of claim 21, wherein said template switchingoligonucleotide comprises at least one or two ribonucleotide residuesand said LNA residue.
 36. The cDNA library of claim 35, wherein said atleast one or two ribonucleotide residues are riboguanine.
 37. The cDNAlibrary of claim 21, wherein said locked nucleic acid residue isselected from the group consisting of locked guanine, locked adenine,locked uracil, locked thymine, locked cytosine, and locked5-methylcytosine.
 38. The cDNA library of claim 21, wherein said lockednucleic acid residue is locked guanine.
 39. The cDNA library of claim21, wherein said locked nucleic acid residue is at the 3′-most position.40. The cDNA library of claim 21, wherein said template switchingoligonucleotide comprises at the 3′-end three nucleotide residuescharacterized by formula rGrG+N, wherein +N represents a lockednucleotide residue.
 41. The cDNA library of claim 40, wherein saidtemplate switching oligonucleotide comprises rGrG+G.
 42. The cDNAlibrary of claim 31, wherein said methyl group donor is betaine, andsaid metal salt is MgCl₂ at a concentration of at least 9 mM.
 43. ThecDNA library of claim 21, wherein the method further comprisesamplifying said DNA strand that is complementary to said RNA moleculeand said template switching oligonucleotide using an oligonucleotideprimer.
 44. The cDNA library of claim 21, wherein said templateswitching oligonucleotide is selected from the oligonucleotides in TableS2.
 45. The cDNA library of claim 21, wherein the cDNA is synthesized onbeads comprising an anchored oligo-dT primer.
 46. The cDNA library ofclaim 45, wherein said oligo-dT primer comprises a sequence of5″-AAGCAGTGGTATCAACGCAGAGTACT₃₀VN-3″, wherein “N” is any nucleosidebase, and “V” is selected from the group consisting of “A”, “C” and “G”.47. The cDNA library of claim 42, wherein the method further comprisesPCR preamplification, tagmentation, and final PCR amplification.
 48. ThecDNA library of claim 47, wherein the PCR preamplification is conductedwithout purifying the cDNA obtained from reverse transcription reaction.49. The cDNA library of claim 21, wherein said RNA is total RNA in acell.